tretinoin and Melanoma

Endometriosis is closely associated with ectopic focal inflammation and immunosuppressive microenvironment. Multiple types of pattern recognition receptors (PRRs) are present in the innate immune system, which are able to detect pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) in both intracellular and external environments. However, the exact role of PRRs in endometriosis and the underlying molecular mechanism are unclear. PRRs are necessary for the innate immune system to identify and destroy invasive foreign infectious agents. Mammals mainly have two types of microbial recognition systems. The first one consists of the membrane-bound receptors, such as toll-like receptors (TLRs), which recognize extracellular microorganisms and activate intracellular signals to stimulate immune responses. The second one consists of the intracellular PRRs, including nod-like receptors (NLRs) and antiviral proteins retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) with helix enzyme domain. In this review, we mainly focus on the key role of PRRs in the pathological processes associated with endometriosis. PRRs recognize PAMPs and can distinguish pathogenic microorganisms from self, triggering receptor ligand reaction followed by the stimulation of host immune response. Activated immune response promotes the transmission of microbial infection signals to the cells. As endometriosis is characterized by dysregulated inflammation and immune response, PRRs may potentially be involved in the activation of endometriosis-associated inflammation and immune disorders. Toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), nod-like receptor family caspase activation and recruitment domain (CARD) domain containing 5 (NLRC5), nod-like receptor family pyrin domain containing 3 (NLRP3), and c-type lectin receptors (CLRs) play essential roles in endometriosis development by regulating immune and inflammatory responses. Absent in melanoma 2 (AIM2)-like receptors (ALRs) and retinoic acid-inducible gene I-like receptors (RLRs) may be involved in the activation of endometriosis-associated immune and inflammation disorders. PRRs, especially TLRs, may serve as potential therapeutic targets for alleviating pain in endometriosis patients. PRRs and their ligands interact with the innate immune system to enhance inflammation in the stromal cells during endometriosis. Thus, tar

Topics: Animals; Carrier Proteins; Endometriosis; Female; Humans; Immunity, Innate; Inflammation; Ligands; Mammals; Melanoma; NLR Proteins; Pathogen-Associated Molecular Pattern Molecules; Receptors, Pattern Recognition; Signal Transduction; Toll-Like Receptors; Tretinoin; Tumor Microenvironment

Preferentially expressed antigen in melanoma (PRAME) is the best characterized member of the PRAME family of leucine-rich repeat (LRR) proteins. Mammalian genomes contain multiple members of the PRAME family whereas in other vertebrate genomes only one PRAME-like LRR protein was identified. PRAME is a cancer/testis antigen that is expressed at very low levels in normal adult tissues except testis but at high levels in a variety of cancer cells. In contrast to most other cancer/testis antigens, PRAME is expressed not only in solid tumors but also in leukemia cells. Expression of PRAME and other members of the PRAME family is regulated epigenetically. PRAME interacts with varying pathways that might be directly involved in the malignant phenotype of cancer cells. For instance, PRAME is able to dominantly repress retinoic acid signaling in these cells. On the other hand, PRAME-derived peptides can be recognized as epitopes by cytotoxic T cells and PRAME represents an attractive target for immunological treatment strategies.

Topics: Animals; Antigens, Neoplasm; Epitopes; Humans; Leucine-Rich Repeat Proteins; Melanoma; Proteins; T-Lymphocytes, Cytotoxic; Tretinoin

This note mechanistically accounts for recent unexplained findings that all-trans retinoic acid (ATRA, also termed tretinoin) exerts an anti-viral effect against hepatitis C virus (HCV) in chronically infected patients, in whom ATRA also showed synergy with interferon-alpha. How HCV replication was suppressed was unclear. Both effects of ATRA can be accounted for by ATRA's upregulation of RIG protein, an 18 kDa product of retinoic induced gene-1. Increased RIG then couples ATRA to increased Type 1 interferons' production. Details of this mechanism predict that ATRA will similarly augment interferon-a activity in treating chronic myelogenous leukemia, melanoma, myeloma and renal cell carcinoma and that the addition of ribavirin and/or bexarotene will each incrementally enhance interferon-a responses in these cancers.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Bexarotene; Carcinoma, Renal Cell; Drug Screening Assays, Antitumor; Hepacivirus; Humans; Interferon-alpha; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Melanoma; Models, Biological; Multiple Myeloma; Ribavirin; Tetrahydronaphthalenes; Treatment Outcome; Tretinoin

Topical tretinoin improves mottling and hyperpigmented lesions of photodamaged skin. The basic mechanisms underlying these effects are not known. It is demonstrated that retinoids inhibit the growth and enhance the differentiation of melanoma cells in vitro, and stimulate the constitutive melanogenesis in melanoma cells in vitro. On the other hand, they inhibit hormonally or pharmacologically induced melanogenesis in these cells. Very few data are available concerning the effect of retinoic acid on normal human melanocytes, but there is some inhibition of growth as in melanoma cells. Retinoic acid appears to have little effect on the melanogenesis of normal human melanocytes grown in vitro using serum-free culture medium. Changes in the shape of these melanocytes suggest that retinoic acid acts on cytoskeleton proteins. Further studies, both in vitro and in vivo, are needed to clarify the effects of retinoic acid on melanocytes.

Topics: Animals; Carrier Proteins; Cell Migration Inhibition; Humans; Melanins; Melanocytes; Melanoma; Mice; Mitosis; Receptors, Retinoic Acid; Skin Aging; Skin Pigmentation; Tretinoin

As described in this review, both partially purified and recombinant interferons are potent modulators of differentiation in diverse cell culture systems (Table 2). Depending on the target cell, interferon exerts either an inhibitory or an inductive effect on cell differentiation. In certain myeloid leukemic cells, such as HL-60, interferon by itself is growth suppressive but does not induce cell maturation, whereas in combination with inducers of differentiation, such as DMSO, TPA or retinoic acid, interferon potentiates their ability to stimulate differentiation in both sensitive and resistant cell populations (Grant et al., 1982, 1983; Tomida et al., 1982). Interferon also interacts synergistically with phorbol ester tumor promoters in inhibiting melanogenesis in murine B-16 cells (Fisher et al., 1981a, 1984a) and adipocyte formation in 3T3 cells (Cioe et al., 1980), whereas the combination is synergistic in inducing differentiation in human melanoma cells (Fisher et al., 1984b,c). In contrast, interferon and TPA display antagonistic effects on differentiation in human skeletal muscle cultures, i.e. interferon induces and TPA inhibits myogenesis (Fisher et al., 1982, 1983). Recent studies have demonstrated the presence of high affinity saturable cell membrane receptors for mouse and human interferons (Aguet, 1980; Branca and Baglioni, 1981, 1982; Mogensen et al., 1981; Branca et al., 1982; Anderson et al., 1982; Joshi et al., 1982; Faltynek et al., 1983; Yonehara et al., 1983; Langer and Pestka, in preparation). Similarly, specific membrane receptors have been identified for phorbol esters and mezerein (Driedger and Blumberg, 1980; Shoyab and Todaro, 1980; Horowitz et al., 1981; Fisher et al., 1981b). These findings suggest that the plasma membrane may be a primary target for mediating the biochemical effects induced by both interferon and phorbol esters. Although the mechanism by which interferon and phorbol esters transmit the necessary membrane signal(s) required for altering differentiation are not known, a possible component of this transmembrane signaling process may involve changes in the physical dynamics of the plasma membrane. It is therefore of interest that both interferon and TPA induce early changes in the fluidity of the plasma membrane (Fisher et al., 1979, 1981b, 1984d; Castagna et al., 1979; Kuhry et al., 1983).(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Adipose Tissue; Animals; Cell Differentiation; Cells, Cultured; Dimethyl Sulfoxide; Hematopoiesis; Humans; Interferons; Leukemia; Leukemia, Erythroblastic, Acute; Melanins; Melanoma; Mice; Muscles; Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin

Topics: Animals; Butyrates; Butyric Acid; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cholecalciferol; Cyclic AMP; Humans; Melanoma; Protein Kinases; Retinoids; Teratoma; Tretinoin; Vitamin A

Topical vitamin A acid (VAA) has various mechanisms of action which may be responsible for its therapeutic success in many different disorders. Although the absorption, metabolism, and excretion of VAA are not completely understood, VAA appears to remain mainly on the skin surface. The question of carcinogenicity is unresolved, and more research is needed to clarify this problem. This article reviews the literature regarding the therapeutic uses of VAA and summarizes various investigators' experiences with VAA.

Topics: Acne Vulgaris; Animals; Callosities; Cocarcinogenesis; Fox-Fordyce Disease; Humans; Ichthyosis; Keloid; Keratoacanthoma; Keratosis; Lichen Planus; Melanoma; Melanosis; Molluscum Contagiosum; Nevus; Psoriasis; Skin Absorption; Skin Diseases; Skin Neoplasms; Tretinoin

Immune checkpoint inhibitors have improved overall survival rates for many cancers, yet the majority of patients do not respond to treatment and succumb to disease progression. One tumor-related mechanism limiting the efficacy of immunotherapies in melanoma is the recruitment and expansion of myeloid-derived suppressor cells (MDSCs). Therefore, targeting MDSCs in combination with immunotherapies is an attractive strategy to improve response rates and effectiveness.. We tested this strategy by designing a randomized phase II clinical trial treating advanced melanoma patients with either Ipilimumab monotherapy or Ipilimumab plus all-trans retinoic acid (ATRA). Clinicaltrails.gov identifier (NCT02403778). The frequency of circulating MDSCs and the activation of CD8(+) T cells was measured by flow cytometry. Expression of immunosuppressive genes was measured with quantitative real time-PCR. T cell suppressive functions were measured by mixed lymphocyte reaction.. Here we show that in vitro treatment with ATRA decreases immunosuppressive function of MDSCs in mixed lymphocyte reactions. Additionally, ATRA reduces the expression of immunosuppressive genes including PD-L1, IL-10, and indoleamine 2,3‑dioxygenase by MDSCs. Furthermore, the addition of ATRA to standard of care Ipilimumab therapy appears safe, as ATRA did not increase the frequency of grade 3 or 4 adverse events. Finally, ATRA significantly decreased the frequency of circulating MDSCs compared to Ipilimumab treatment alone in advanced-stage melanoma patients.. These results illustrate the importance of MDSCs in immunotherapy resistance and provide evidence that targeting MDSCs in cancer patients may augment immunotherapeutic approaches.

Topics: Adult; Aged; Antineoplastic Agents, Immunological; Antineoplastic Combined Chemotherapy Protocols; Female; Humans; Ipilimumab; Male; Melanoma; Middle Aged; Myeloid-Derived Suppressor Cells; Tretinoin

Atypical naevi are potential precursors of melanoma and markers of increased melanoma risk. To examine the possibility of chemoprevention of melanoma by retinoids, we studied the effect of topical tretinoin 0.1% (all-transretinoic acid; vitamin A acid) and tretinoin 0.1% with hydrocortisone on atypical naevi. Thirty patients with atypical naevi were enrolled in a prospective randomized double blind study. For each patient three comparable naevi were selected and randomized to receive tretinoin 0.1% (T), tretinoin 0.1% with hydrocortisone 1% (C) or a placebo cream (P) once a week under Actiderm occlusion for 4 months. Baseline views of the naevi, taken with a videomicroscope (magnification 20 x), were assessed for morphological changes compared with views taken 2 months after the beginning of treatment, 1 week after completion of treatment and 6 months later. After completion of the study all naevi in the T and C groups and six naevi in the P group were removed and evaluated histologically for the presence of atypia. The number of naevi that had changed in colour or size was significantly higher in the T and C groups compared with the placebo group. A size reduction took place in 42.9% (T) and 40.0% (C) of the naevi and the colour changed in 75.0% (T) and 66.7% (C). The effect of treatment, in general subtle, did not differ significantly between groups T and C, but naevi treated with C became significantly less irritated. Histologically, 75.0% of the naevi treated with T and 69.6% of the naevi treated with C were atypical. Therefore, no major change was seen in the clinical aspect of atypical naevi after treatment with tretinoin 0.1% or tretinoin with hydrocortisone 1%, and most of the treated naevi still met the histological criteria for atypia after the treatment period. The current management of follow-up of atypical naevi and excision when change to melanoma is suspected is therefore still recommended. Nevertheless, some response was seen, which may justify a further exploration of tretinoin and hydrocortisone 1% therapy for a longer treatment period in combination with research to clarify its mechanism.

Topics: Adult; Antineoplastic Agents; Female; Humans; Hydrocortisone; Male; Melanoma; Middle Aged; Nevus; Occlusive Dressings; Pigmentation; Placebos; Prospective Studies; Skin Neoplasms; Tretinoin

Laboratory evidence of disseminated intravascular coagulation (DIC) and/or fibrinolysis is present in the majority of patients with acute promyelocytic leukemia (APL). Historically, early hemorrhagic death (EHD) occurred in 10% to 30% of patients treated with chemotherapy. All-trans retinoic acid (ATRA), a differentiating agent, has a CR rate above 80% in patients, with ATRA-associated leukocytosis. We studied thrombotic events in this population and compared it to patients treated with chemotherapy alone. The results of studies using ATRA in patients with APL were reviewed. Patients received ATRA 45-50 mg/m(2) orally in two divided doses daily until complete remission. In newly diagnosed patients, Idarubicin 12 mg/m(2)/day was given intravenously for 4 to 5 days beginning on the fifth day of ATRA therapy or when the white blood cell count (WBC) was over 10x 10(3)/mu l. Thrombotic complications were noted in 3 of 31 patients during induction. Two died from thrombotic events during therapy with multiple thromboses documented at autopsy. ATRA syndrome was suspected in 2 of the patients with thromboses and only 1 of the patients without thrombosis. In previous studies, 1 of 25 APL patients treated with chemotherapy alone had thrombotic events during therapy. In conclusion, treatment of APL with ATRA may decrease the incidence of hemorrhagic complications, but does not eliminate thrombosis. While thrombotic events were not significantly increased in patients treated with ATRA, they were more common in patients suspected of having ATRA syndrome.

Topics: Adult; Amsacrine; Antibiotics, Antineoplastic; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cerebral Hemorrhage; Cohort Studies; Cytarabine; Disseminated Intravascular Coagulation; Fatal Outcome; Female; Germinoma; Hemorrhage; Humans; Idarubicin; Incidence; Infarction; Leukemia, Promyelocytic, Acute; Leukocytosis; Male; Melanoma; Middle Aged; Myocardial Infarction; Neoplasms, Multiple Primary; Remission Induction; Retrospective Studies; Spleen; Thrombosis; Treatment Outcome; Tretinoin

As potential precursors of melanoma and markers of increased melanoma risk, dysplastic nevi are suitable targets of strategies for melanoma chemoprevention. We report the results of a pilot study of topical retinoic acid in patients with dysplastic nevi.. Five male patients with dysplastic nevi applied tretinoin to half of the back for 6 months. Baseline photographs of dysplastic nevi were compared with posttreatment photographs and assessed for morphologic change. At study completion, each subject had four nevi excised from the treated side and four from the untreated side of the back. Biopsies were histologically evaluated for the presence of dysplasia.. All patients developed signs of irritation as a result of treatment. One patient was not compliant with treatment due to skin irritation. The four compliant patients showed significant decreases in the clinical atypia of treated lesions, with concomitant fading and even disappearance of many treated nevi. Histologically, only four of 16 treated nevi met histologic criteria for dysplasia, in comparison to 13 of 16 untreated nevi.. These results suggest that there is concomitant clinical and histologic improvement in a significant percentage of dysplastic nevi treated with topical tretinoin. However, the utility of topical tretinoin for chemoprevention of melanoma is limited by difficulty of application and associated inflammation. While new strategies in chemoprevention of melanoma are explored, sun protection and assiduous avoidance of sunburn must remain the mainstay of melanoma prevention.

Topics: Administration, Topical; Adult; Anticarcinogenic Agents; Cell Differentiation; Dysplastic Nevus Syndrome; Humans; Immunohistochemistry; Male; Melanoma; Pilot Projects; Tretinoin

Retinoids, the natural and synthetic analogs of vitamin A, are growth-inhibiting and differentiation-inducing agents and show clinical promise as chemopreventive and antineoplastic agents. Fenretinide, a new synthetic retinoid, has antitumor activity in certain in vitro and in vivo model systems and was relatively nontoxic in phase I trials. Based on these data, we designed a phase II study of Fenretinide involving 31 patients with advanced breast cancer [15] and melanoma [16], two cancers shown to be responsive to this agent in preclinical models. Fenretinide was inactive in patients with advanced disease. Toxicity was mild, and reversible. Mucocutaneous side effects occurred in 16 (52%) patients. Nyctalopia developed in three patients one of whom developed decreased B-wave amplitude of the scotopic electroretinogram. The minimal toxicity and significant activity in preclinical studies make this an attractive agent for future breast cancer chemoprevention studies.

Topics: Adult; Aged; Breast Neoplasms; Drug Evaluation; Female; Fenretinide; Humans; Male; Melanoma; Middle Aged; Tretinoin

The retinoids have been investigated extensively as chemopreventive and therapeutic agents in a variety of neoplasms. They have been shown to inhibit the proliferation of transformed cell lines in vitro and transplanted tumors in vivo. In cultured murine melanoma cells, retinoids inhibit proliferation and induce differentiation. Human melanoma cell lines have shown a mixed response. The clinical experience with retinoids in melanoma has been limited. Previously we investigated the activity of topical B-all-trans-retinoic acid (Retin-A, vitamin A acid, retinoic acid, and tretinoin) against intracutaneous metastases from malignant melanoma. We saw complete remission of multiple lesions in one individual and regression of several lesions in a second patient. This experience led us to conduct the present pilot trial of topical tretinoin in dysplastic nevus syndrome. The latter is a precursor of malignant melanoma. We saw regression of some of the treated lesions to benign nevi showing minimal or no dysplasia. Thus topical tretinoin appears to possess some activity against melanoma and at least one of its precursor conditions. In view of these preliminary results, more extensive trials are warranted to better define the role of tretinoin in the chemoprevention of malignant melanoma in high-risk lesions.

Topics: Administration, Topical; Adolescent; Clinical Trials as Topic; Dysplastic Nevus Syndrome; Humans; Male; Melanoma; Middle Aged; Pilot Projects; Skin Neoplasms; Tretinoin

A phase Ib/II clinical trial was conducted to evaluate the safety and efficacy of the combination of all-trans retinoic acid (ATRA) with pembrolizumab in patients with stage IV melanoma.. Anti-PD-1 naïve patients with stage IV melanoma were treated with pembrolizumab plus supplemental ATRA for three days surrounding each of the first four pembrolizumab infusions. The primary objective was to establish the MTD and recommended phase II dose (RP2D) of the combination. The secondary objectives were to describe the safety and toxicity of the combined treatment and to assess antitumor activity in terms of (i) the reduction in circulating myeloid-derived suppressor cell (MDSC) frequency and (ii) progression-free survival (PFS).. Twenty-four patients were enrolled, 46% diagnosed with M1a and 29% with M1c stage disease at enrollment. All patients had an ECOG status ≤1, and 75% had received no prior therapies. The combination was well tolerated, with the most common ATRA-related adverse events being headache, fatigue, and nausea. The RP2D was established at 150 mg/m2 ATRA + 200 mg Q3W pembrolizumab. Median PFS was 20.3 months, and the overall response rate was 71%, with 50% of patients experiencing a complete response, and the 1-year overall survival was 80%. The combination effectively lowered the frequency of circulating MDSCs.. With a favorable tolerability and high response rate, this combination is a promising frontline treatment strategy for advanced melanoma. Targeting MDSCs remains an attractive mechanism to enhance the efficacy of immunotherapies, and this combination merits further investigation. See related commentary by Olson and Luke, p. 1167.

Topics: Antineoplastic Combined Chemotherapy Protocols; Humans; Melanoma; Myeloid-Derived Suppressor Cells; Neoplasms, Second Primary; Tretinoin

Myeloid-derived suppressor cells (MDSC) are associated with resistance to anti-PD-1 therapies. All-trans retinoic acid (ATRA) may induce maturation of MDSCs and alter their immunosuppressive effects. Adding ATRA to pembrolizumab may target this resistance mechanism to enhance the overall impact of anti-PD-1-based immunotherapy. See related article by Tobin et al., p. 1209.

Topics: Cell Differentiation; Humans; Melanoma; Myeloid-Derived Suppressor Cells; Tretinoin

Melanoma originates from the malignant transformation of melanocytes. Compared with other skin cancers, melanoma has a higher fatality rate. The 5-year survival rate of patients with early-stage primary melanoma through surgical resection can reach more than 90%. However, the 5-year survival rate of patients with metastatic melanoma is only 25%. Therefore, accurate assessment of melanoma progression is critical. Previous studies have found that Retinoic Acid Induced 14(RAI14) is critical in tumorigenesis. However, the biological function of RAI14 for the development of melanoma is unclear. In this study, RAI14 is highly expressed in melanoma and correlated with prognosis. The expression of RAI14 can affect the proliferation, migration and invasion of melanoma cells. F-Box Protein 32(FBXO32) is an E3 ubiquitin ligase of c-MYC. We found that RAI14 affects the transcriptional expression of FBXO32 and regulates the stability of c-MYC. These results suggest that RAI14 play an important role in the growth of melanoma and is expected to be a therapeutic target for melanoma.

Topics: Cell Proliferation; Cell Transformation, Neoplastic; Cytoskeletal Proteins; F-Box Proteins; Humans; Melanoma; Muscle Proteins; Proto-Oncogene Proteins c-myc; Skin Neoplasms; SKP Cullin F-Box Protein Ligases; Transcription Factors; Tretinoin; Ubiquitin-Protein Ligases

Embryonic stem cells (ESCs) present a characteristic pluripotency heterogeneity correspondent to specific metastates. We recently demonstrated that retinoic acid (RA) induces an increase in a specific 2C-like metastate marked by target genes specific to the two-cell embryo stage in preimplantation. Prame (Preferentially expressed antigen in melanoma) is one of the principal actors of the pluripotency stage with a specific role in RA responsiveness. Additionally, PRAME is overexpressed in a variety of cancers, but its molecular functions are poorly understood. To further investigate Prame's downstream targets, we used a chromatin immunoprecipitation sequencing (ChIP-seq) assay in RA-enriched 2C-like metastates and identified two specific target genes,

Topics: Antigens, Neoplasm; Cyclin-Dependent Kinase 8; Embryonic Stem Cells; Humans; Melanoma; Tretinoin

Surgical excision is standard-of-care for primary invasive melanoma, but best care can be unclear for patients who are surgically high-risk or for whom resection may be excessively morbid. Alternatives to surgical excision have emerged for treatment of metastatic melanoma but have not yet been explored for primary invasive melanoma. Two elderly patients with primary invasive melanoma with many medical co-morbidities who were not surgical candidates were determined to be appropriate candidates for an intralesional IL-2 based regimen. Herein we report their clinical and histological outcome. An intralesional-based regimen (intralesional IL-2, topical imiquimod cream 5%, and tretinoin cream 0.1% under occlusion to the treatment site) was administered over the course of six to seven weeks, followed by two weeks of topical-only therapy. A complete response was seen after eight to nine weeks of treating invasive melanomas that were ≥1.85 mm and 5.5 mm thick. For patients with primary invasive melanoma on high morbidity sites and patients who are poor surgical candidates, a neoadjuvant intralesional IL-2-based approach may be a reasonable alternative. The two cases presented here suggest that alternative intralesional-based treatment modalities may minimize the size of the excision site and can be associated with complete histological clearance of invasive melanoma.

Topics: Aged; Aminoquinolines; Antineoplastic Agents; Humans; Imiquimod; Melanoma; Skin Neoplasms; Treatment Outcome; Tretinoin

Topics: Administration, Topical; Aminoquinolines; Antineoplastic Agents; Humans; Imiquimod; Melanoma; Skin Neoplasms; Tretinoin

Malignant melanoma is a common form of skin cancer that contains different cell types recognized by various cell surface markers. Dacarbazine-based combination chemotherapy is frequently used for the treatment of melanoma. Despite its potent anticancer properties, resistance to dacarbazine develops in malignant melanoma. Here, we aim to improve response to dacarbazine therapy by pretreatment with all-trans retinoic acid (ATRA) in CD117. Collectively, combined treatment with ATRA and dacarbazine induced more apoptosis and enhanced the cell cycle arrest of CD117

Topics: Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dacarbazine; Drug Synergism; G1 Phase; Humans; Melanoma; Melanoma, Cutaneous Malignant; Proto-Oncogene Proteins c-kit; Resting Phase, Cell Cycle; Skin Neoplasms; Tretinoin

PLX4032 is commonly used in the treatment of advanced melanoma patients with BRAF-V600E mutation. The aim of this study was to elucidate the mechanisms by which up-regulation of PIN1 confers PLX4032 resistance in melanoma.. The expression of PIN1 as well as the cytotoxic effects of combinatorial treatment of PLX4032 and all-trans retinoic acid (ATRA) were investigated by immunoblotting, MTT assay, TUNEL assay, and soft agar assay.. PIN1 expression is up-regulated in A375R cells, a PLX4032-resistant subline of melanoma cells generated from an A375 cell line, compared to parental A375 cells. Indeed, PIN1 positively regulated the expression of EGFR in A375R cells and led to activation of the RAF/MEK/ERK pathway. Importantly, PLX4032, when used in combination with ATRA, an inhibitor of PIN1, reduced EGFR expression, and consequently reduced cell viability and anchorage-independent growth of A375R cells compared to PLX4032 alone. Furthermore, co-treatment with ATRA and PLX4032 increased cleaved PARP and DNA fragmentation in A375R cells.. PIN1 plays an important role in the development of PLX4032 resistance through up-regulation of EGFR expression.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Survival; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Male; Melanoma; Mice; NIMA-Interacting Peptidylprolyl Isomerase; Tretinoin; Vemurafenib; Xenograft Model Antitumor Assays

Dysregulation of retinoic acid signaling is implicated in several human cancer types, including melanoma where the gene encoding retinoic acid receptor beta (RARβ) is frequently silenced by promoter hypermethylation. In this chapter, we describe some of the experimental procedures that we have used to characterize the role of RARβ signaling on the regulation of cellular metabolism in melanoma. Central to these studies is the use of the Seahorse XF Analyzer, which allows real-time assessment of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in cultured cells as readouts for oxidative phosphorylation and glycolysis, respectively. The levels of RARβ signaling can be modulated using RARβ agonists (e.g., all-trans retinoic acid) and antagonists (e.g., LE135). The bioenergetic profiles of melanoma cells in response to RARβ modulators and other metabolic modifiers can be the basis for defining new therapeutic strategies.

Topics: Animals; Cell Culture Techniques; Cell Line, Tumor; Dibenzazepines; Glycolysis; Humans; Melanoma; Metabolomics; Mitochondria; Oxidative Phosphorylation; Oxygen Consumption; Phenotype; Receptors, Retinoic Acid; Signal Transduction; Smegmamorpha; Tretinoin

Malignant melanoma is a highly lethal disease, and advanced stages of melanoma have proven to be resistant to many chemotherapeutic drugs including temozolomide and paclitaxel. Cancer stem cells (CSCs) have been identified and isolated in different cancers including melanoma, and have been proven playing important role in the drug resistance. Retinoic acid (RA) is a promising anticancer agent, which can induce differentiation of CSCs. The main purpose of the present study was to evaluate the possible RA-induced differentiation of melanoma CSCs and sensitization of melanoma CSCs to paclitaxel. Our results show that CSCs of human melanoma A375 cells was more tolerant to paclitaxel than other non-CSCs melanoma cells. On the contrary, RA had stronger inhibitory effect on melanoma CSCs than on non-CSCs. At the same time, RA could arrest the cell cycle of CSCs and reduce the expression of Sox-2, Oct-4 in CSCs of melanoma, thereby induced the differentiation of CSCs and increased its sensitivity to paclitaxel. With this study we concluded that RA increases the anticancer effect of paclitaxel by inducing differentiation of cancer stem cells in melanoma, and the combined application of RA and paclitaxel may be more effective in the treatment of melanoma.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Neoplastic Stem Cells; Paclitaxel; SOXB1 Transcription Factors; Tretinoin

All-trans-retinoic acid (ATRA) is a differentiating agent used in the treatment of acute-promyelocytic-leukemia (APL) and it is under-exploited in other malignancies despite its low systemic toxicity. A rational/personalized use of ATRA requires the development of predictive tools allowing identification of sensitive cancer types and responsive individuals.. RNA-sequencing data for 10 080 patients and 33 different tumor types were derived from the TCGA and Leucegene datasets and completely re-processed. The study was carried out using machine learning methods and network analysis.. We profiled a large panel of breast-cancer cell-lines for in vitro sensitivity to ATRA and exploited the associated basal gene-expression data to initially generate a model predicting ATRA-sensitivity in this disease. Starting from these results and using a network-guided approach, we developed a generalized model (ATRA-21) whose validity extends to tumor types other than breast cancer. ATRA-21 predictions correlate with experimentally determined sensitivity in a large panel of cell-lines representative of numerous tumor types. In patients, ATRA-21 correctly identifies APL as the most sensitive acute-myelogenous-leukemia subtype and indicates that uveal-melanoma and low-grade glioma are top-ranking diseases as for average predicted responsiveness to ATRA. There is a consistent number of tumor types for which higher ATRA-21 predictions are associated with better outcomes.. In summary, we generated a tumor-type independent ATRA-sensitivity predictor which consists of a restricted number of genes and has the potential to be applied in the clinics. Identification of the tumor types that are likely to be generally sensitive to the action of ATRA paves the way to the design of clinical studies in the context of these diseases. In addition, ATRA-21 may represent an important diagnostic tool for the selection of individual patients who may benefit from ATRA-based therapeutic strategies also in tumors characterized by lower average sensitivity.

Topics: Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Female; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; Glioma; Humans; Leukemia, Promyelocytic, Acute; Machine Learning; Melanoma; Models, Theoretical; Sequence Analysis, RNA; Tretinoin; Uveal Neoplasms

Vemurafenib is a selected BRAF kinase inhibitor approved for treating metastatic or unresectable melanoma, which has numerous cutaneous side effects unfortunately, including three previously reported cases of asymptomatic areola and/or nipple hyperkeratosis. We present the first case of painful bilateral nipple hyperkeratosis secondary to vemurafenib in an 84-year-old woman. She was successfully treated with tretinoin 0.05% cream that allowed her to comfortably continue treatment. With increased awareness of this condition, we found a second case of asymptomatic nipple hyperkeratosis secondary to vemurafenib in our clinic. As this medication gains acceptance for treatment of metastatic melanoma, it is imperative that dermatologists are aware of this potentially uncomfortable side effect that can result in decreased compliance and impaired quality of life.

Topics: Aged, 80 and over; Antineoplastic Agents; Female; Humans; Indoles; Keratosis; Melanoma; Nipples; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Quality of Life; Skin Neoplasms; Sulfonamides; Tretinoin; Vemurafenib

BRAF-mutant melanoma is characterized by aggressive metastatic potential and therapeutic resistance. The innate immune receptor RIG-I has emerged as a potential target in melanoma therapies but the contributing pathways involved in anti-cancer activity are poorly characterized.. Baseline and ATRA-induced expression of RIG-I in nine (3 wild type and 6 BRAF-mutant) melanoma cell lines was measured with Q-PCR and Western blot. Ligand-specific stimulation of RIG-I was detected by Q-PCR and ELISA. Activation of the RIG-I-coupled IRF3, NF-κB and MAPK pathways was tested with protein array and Western blot. Cell proliferation and apoptosis was monitored by flow cytometry and cell counting. Down modulation of MKP-1 expression in melanoma cells was performed by specific siRNA.. Short-term ATRA pre-treatment increases the expression of RIG-I in BRAF-mutant melanoma cells. Specific activation of RIG-I by 5'ppp-dsRNA leads to increased activity of the IRF3-IFNβ pathway but does not influence NF-κB signaling. RIG-I mediates the targeted dephosphorylation of several MAPKs (p38, RSK1, GSK-3α/β, HSP27) via the endogenous regulator MKP-1 resulting in decreased melanoma cell proliferation.. RIG-I has the potential to exert anticancer activity in BRAF-mutant melanoma via controlling IFNβ production and MAPK signaling. This is the first study showing that RIG-I activation results in MKP-1-mediated inhibition of cell proliferation via controlling the p38-HSP27, c-Jun and rpS6 pathways thus identifying RIG-I and MKP-1 as novel and promising therapeutical targets.

Topics: Cell Line, Tumor; Cell Proliferation; Cytokines; DEAD Box Protein 58; Dual Specificity Phosphatase 1; Humans; Interferon Regulatory Factor-3; MAP Kinase Signaling System; Melanoma; Mitogen-Activated Protein Kinases; NF-kappa B; Proto-Oncogene Proteins B-raf; Receptors, Immunologic; Tretinoin

BACKGROUND The identification and use of novel compounds alone or in combination hold promise for the fight against NRAS mutant melanoma. MATERIAL AND METHODS We screened a kinase-specific inhibitor library through combining it with α-Mangostin in NRAS mutant melanoma cell line, and verified the enhancing effect of α-Mangostin through inhibition of the tumorigenesis pathway. RESULTS Within the kinase inhibitors, retinoic acid showed a significant synergistic effect with α-Mangostin. α-Mangostin also can reverse the drug resistance of retinoic acid in RARa siRNA-transduced sk-mel-2 cells. Colony assay, TUNEL staining, and the expressions of several apoptosis-related genes revealed that a-Mangostin enhanced the effect of retinoic acid-induced apoptosis. The combination treatment resulted in marked induction of ROS generation and inhibition of the AKT/S6 pathway. CONCLUSIONS These results indicate that the combination of these novel natural agents with retinoid acid may be clinically effective in NRAS mutant melanoma.

Topics: Apoptosis; Biological Products; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; GTP Phosphohydrolases; Humans; Melanoma; Membrane Proteins; Mutation; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Reproducibility of Results; Signal Transduction; Tretinoin; Xanthones

Melanoma is a cancer that arises from melanocytes, specialized pigmented cells that are found predominantly in the skin. The incidence of malignant melanoma has significantly increased over the last decade. With the development of therapy, the survival rate of some kind of cancer has been improved greatly. But the treatment of melanoma remains unsatisfactory. Much of melanoma's resistance to traditional chemotherapy is believed to arise intrinsically, by virtue of potent growth and cell survival-promoting genetic alteration. Therefore, significant attention has recently been focused on differentiation therapy, as well as differentiation inducer compounds. In previous study, we found isoliquiritigenin (ISL), a natural product extracted from licorice, could induce B16F0 melanoma cell differentiation. Here we investigated the transcriptional response of melanoma differentiation process induced by ISL and all-trans-retinoic acid (RA). Results showed that 390 genes involves in 201 biochemical pathways were differentially expressed in ISL treatment and 304 genes in 193 pathways in RA treatment. Differential expressed genes (DGEs, fold-change (FC)≥10) with the function of anti-proliferative and differentiation inducing indicated a loss of grade malignancy characteristic. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated glutathione metabolism, glycolysis/gluconeogenesis and pentose phosphate pathway were the top three relative pathway perturbed by ISL, and mitogen-activated protein kinase (MAPK) signaling pathway was the most important pathway in RA treatment. In the analysis of hierarchical clustering of DEGs, we discovered 72 DEGs involved in the process of drug action. We thought Cited1, Tgm2, Xaf1, Cd59a, Fbxo2, Adh7 may have critical role in the differentiation of melanoma. The evidence displayed herein confirms the critical role of reactive oxygen species (ROS) in melanoma pathobiology and provides evidence for future targets in the development of next-generation biomarkers and therapeutics.

Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Chalcones; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; MAP Kinase Signaling System; Melanoma; Pentose Phosphate Pathway; Transcriptome; Tretinoin

Topics: Aged; Alitretinoin; Antineoplastic Agents; Female; Forearm; Humans; Indoles; Keratoacanthoma; Melanoma; Proto-Oncogene Proteins B-raf; Retinoids; Skin Neoplasms; Sulfonamides; Tretinoin; Vemurafenib

Topics: Antigens, Neoplasm; Cell Line, Tumor; Enhancer of Zeste Homolog 2 Protein; Humans; Melanoma; Polycomb Repressive Complex 2; Prostaglandin D2; Tretinoin

Retinoids such as all-trans retinoic acid (ATRA) influence cell growth, differentiation and apoptosis and may play decisive roles in tumor development and progression. An essential retinoid-metabolizing enzyme known as lecithin retinol acyltransferase (LRAT) is expressed in melanoma cells but not in melanocytes catalysing the esterification of all-trans retinol (ATRol). In this study, we show that a stable LRAT knockdown (KD) in the human melanoma cell line SkMel23 leads to significantly increased levels of the substrate ATRol and biologically active ATRA. LRAT KD restored cellular sensitivity to retinoids analysed in cell culture assays and melanoma 3D skin models. Furthermore, ATRA-induced gene regulatory mechanisms drive depletion of added ATRol in LRAT KD cells. PCR analysis revealed a significant upregulation of retinoid-regulated genes such as CYP26A1 and STRA6 in LRAT KD cells, suggesting their possible involvement in mediating retinoid resistance in melanoma cells. In conclusion, LRAT seems to be important for melanoma progression. We propose that reduction in ATRol levels in melanoma cells by LRAT leads to a disturbance in cellular retinoid level. Balanced LRAT expression and activity may provide protection against melanoma development and progression. Pharmacological inhibition of LRAT activity could be a promising strategy for overcoming retinoid insensitivity in human melanoma cells.

Topics: Acyltransferases; Catalysis; Cell Line, Tumor; Cells, Cultured; Disease Progression; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Keratinocytes; Melanocytes; Melanoma; Melanoma, Cutaneous Malignant; Skin Neoplasms; Tretinoin; Vitamin A

Aldehyde dehydrogenase 1A1 (ALDH1A1), an enzyme that catalyses the conversion of lipid aldehydes to lipid carboxylic acids, plays pleiotropic roles in UV-radiation resistance, melanogenesis and stem cell maintenance. In this study, a combination of RNAi and pharmacologic approaches were used to determine which ALDH1A1 substrates and products regulate melanogenesis. Initial studies revealed that neither the UV-induced lipid aldehyde 4-hydroxy-2-nonenal nor the ALDH1A1 product all-trans retinoic acid appreciably induced melanogenesis. In contrast, both the ALDH1A1 substrate 9-cis retinal and its corresponding product 9-cis retinoic acid potently induced the accumulation of MITF mRNA, Tyrosinase mRNA and melanin. ALDH1A1 depletion inhibited the ability of 9-cis retinal but not 9-cis retinoic acid to stimulate melanogenesis, indicating that ALDH1A1 regulates melanogenesis by catalysing the conversion of 9-cis retinal to 9-cis retinoic acid. The addition of potent ALDH1A inhibitors (cyanamide or Angeli's salt) suppressed Tyrosinase and MITF mRNA accumulation in vitro and also melanin accumulation in skin equivalents, suggesting that 9-cis retinoids regulate melanogenesis in the intact epidermis. Taken together, these studies not only identify cyanamide as a potential novel treatment for hyperpigmentary disorders, but also identify 9-cis retinoic acid as a pigment stimulatory agent that may have clinical utility in the treatment of hypopigmentary disorders, such as vitiligo.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Alitretinoin; Cell Line, Tumor; Cells, Cultured; Cyanamide; Humans; Melanins; Melanocytes; Melanoma; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Retinal Dehydrogenase; Skin; Skin Neoplasms; Skin Pigmentation; Tretinoin

Melanoma incidence is dramatically increasing and the available treatments beyond partial efficacy have severe side effects. Retinoids are promising anticancer agents, but their clinical use has been limited by their toxicity, although a combination with other agents can possibly generate a therapeutic action at lower dosage. Thus, we investigated the effects of all-trans-retinoic acid combined with the antiestrogen endoxifen on melanoma cell proliferation and the effects were compared with its pro-drug tamoxifen. Moreover, we evaluated the effects of these combinations on non-neoplasic cells and assessed mitochondrial bioenergetic functions, to predict their potential toxicity. Individually, all-trans-retinoic acid and the antiestrogens endoxifen and tamoxifen decreased melanoma cell biomass, cell viability and DNA synthesis, without increased cell death, suggesting that the compounds inhibited cell proliferation. Noteworthy, endoxifen decreased cell proliferation more efficiently than tamoxifen. The combination of endoxifen with all-trans-retinoic acid enhanced the antiproliferative effects of the compounds individually more potently than tamoxifen, which did not enhance the effects induced by all-trans-retinoic acid alone, and blocked cell cycle progression in G1. Moreover, the combination of all-trans-retinoic acid with endoxifen significantly decreased melanoma cells migration, whereas the combination with tamoxifen did not present significant effects. At the concentrations used the compounds did not induce cytotoxicity in non-neoplasic cells and liver mitochondrial bioenergetic function was not affected. Altogether, our results show for the first time that a combined treatment of all-trans-retinoic acid with endoxifen may provide an anti-proliferative and anti-migration effect upon melanoma cells without major toxicity, offering a powerful therapeutic strategy for malignant melanoma.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Movement; Cell Proliferation; Energy Metabolism; Estrogen Receptor Modulators; Female; Liver; Male; Melanoma; Mice; Mitochondria; Rats; Tamoxifen; Tretinoin; Xenograft Model Antitumor Assays

Melanoma genomes contain thousands of alterations including: mutations, copy number alterations, structural aberrations, and methylation changes. The bulk of this variation is stochastic and functionally neutral, with only a small minority representing "drivers" that contribute to the genesis and maintenance of tumors. Drivers are often directly or inversely correlated across tumors, reflecting the molecular and regulatory signaling pathways in which they operate. Here, a profile of genetic and epigenetic drivers in 110 human melanoma cell lines was generated and searched for non-random distribution patterns. Statistically significant mutual exclusivity was revealed among components of each of the p16(INK4A)-CDK4-RB, RAS-RAF-MEK-ERK and PI3K-AKT signaling pathways. In addition, an inverse correlation was observed between promoter hypermethylation of retinoic acid receptor β (RARB) and CDKN2A alterations affecting p14(ARF) (P < 0.0001), suggesting a functional link between RARβ signaling and the melanoma-suppressive activities of p14(ARF). Mechanistically, all-trans retinoic acid (ATRA) treatment increased the expression of p14(ARF) in primary human melanocytes and the steady-state levels of p14(ARF) in these cells were shown to be regulated via RARβ. Furthermore, the ability of ATRA to induce senescence is reduced in p14(ARF)-depleted melanocytes, and we provide proof-of-concept that ATRA can induce irreversible growth arrest in melanoma cells with an intact RARβ-p14(ARF) signaling axis, independent of p16(INK4A) and p53 status.. These data highlight the power of mutual exclusivity analysis of cancer drivers to unravel molecular pathways and establish a previously unrecognized cross-talk between RARβ and p14(ARF) with potential implications for melanoma treatment.

Topics: Cell Line, Tumor; Epigenesis, Genetic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Receptors, Retinoic Acid; Signal Transduction; Tretinoin; Tumor Suppressor Protein p14ARF

Disturbance in vitamin A metabolism seems to be an important attribute of cancer cells. Retinoids, particularly retinoic acid, have critical regulatory functions and appear to modulate tumor development and progression. The key step of vitamin A metabolism is the esterification of all-trans retinol, catalyzed by lecithin/retinol acyltransferase (LRAT). In this work, we show that malignant melanoma cells are able to esterify all-trans retinol and subsequently isomerize all-trans retinyl esters (RE) into 11-cis retinol, whereas their benign counterparts-melanocytes are not able to catalyze these reactions. Besides, melanoma cell lines express lecithin/retinol acyltranseferase both at the mRNA and protein levels. In contrast, melanocytes do not express this enzyme at the protein level, but mRNA of lecithin/retinol acyltransefrase could still be present at mRNA level. RPE65 is expressed in both melanocytic counterparts, and could be involved in the subsequent isomerization of RE produced by lecithin/retinol acyltransefrase to 11-cis retinol. Cellular retinol-binding protein 2 does not appear to be involved in the regulation of all-trans retinol esterification in these cells. Expression of LRAT and RPE65 can be modulated by retinoids. We propose that the post-transcriptional regulation of lecithin/retinol acyltransefrase could be involved in the differential expression of this enzyme. Besides, activities of LRAT and RPE65 may be important for removal of all-trans retinal which is the substrate for retinoic acid production in skin cells. Consequently, the decreasing cellular amount of retinoic acid and its precursor molecules could result in a change of gene regulation.

Topics: Acyltransferases; Carrier Proteins; Cell Line, Tumor; cis-trans-Isomerases; Eye Proteins; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Melanocytes; Melanoma; Receptors, Retinoic Acid; Retinoid X Receptors; Retinol-Binding Proteins, Cellular; RNA Interference; RNA, Messenger; Tretinoin; Vitamin A

The effects of six lipophilic vitamins: tretinoin (ATRA), vitamin D(3) (VD(3)), VE, VK(1), VK(3), and VK(5) on cell proliferation and apoptosis in human A375 melanoma cells were investigated. VD(3), VK(3), and VK(5) were found to inhibit cell proliferation significantly at concentration ranges of 10-100 μmol/L (p<0.01), while the other vitamins did not show inhibitory effects at 100 μmol/L. VK(3) and VK(5) showed the strongest effects with IC(50) values of less than 10 μmol/L. Dacarbazine slightly inhibited the proliferation of A375 cells at a concentration range of 25-100 μmol/L, but the effects were not statistically significant. VK(3) and VK(5) increased annexin-V positive apoptotic cells, as well as activating caspase-3, in A375 cells. Our findings showed that VD(3), VK(3,) and VK(5) inhibited the growth of dacarbazine resistant human melanoma cells, while ATRA, VE, and VK(1) had little effect on the cell growth. The effects of VK(3) and VK(5) were observed at concentrations lower than 10 μmol/L, which are suggested to have resulted from apoptosis-induction in the melanoma cells.

Topics: Annexin A5; Antineoplastic Agents; Apoptosis; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cholecalciferol; Dacarbazine; Drug Resistance, Neoplasm; Humans; Hydrophobic and Hydrophilic Interactions; Inhibitory Concentration 50; Melanoma; Tretinoin; Vitamin K; Vitamins

In the last three decades, the incidence of melanoma has increased worldwide and no effective treatment modalities have been developed yet. All-trans retinoic acid (ATRA) and polyinosinic:polycytidylic acid (polyI:C) are strong inducers of toll-like receptor 3 (TLR3) and MDA5 expression, and polyI:C-induced TLR3 and MDA5 signaling specifically causes cell death in melanoma cells in vitro. We addressed the question of whether ATRA pretreatment could enhance the efficacy of polyI:C and, if so, would ATRA have any additional effects on this process. We found that the combined treatment of human melanoma cells with ATRA and polyI:C strongly increased the expression of TLR3 and MDA5 in both WM35 and WM983A cells associated with significantly higher mRNA and secreted levels of interferon β (IFNβ), CXCL1, CXCL8/IL-8, CXCL9, and CXCL10 than cells treated with either ATRA or polyI:C. Silencing of MDA5 by siRNA moderately affected IFNβ secretion, whereas TLR3 knockdown interfered with both CXCL chemokine and IFNβ production. Furthermore, the supernatants of ATRA+polyI:C-activated cultures increased the migration of both human monocyte-derived macrophages and CD1a dendritic cells significantly as compared with the supernatants of cells treated with either ATRA or polyI:C, and this effect occurred in a TLR3-dependent manner. In conclusion, consecutive treatment with ATRA and polyI:C results in strong, TLR3/MDA5-mediated chemokine and IFN responses in cultured human melanoma cells, which triggers a functional migratory response in professional antigen-presenting cells. This novel mode of concomitant activation may represent a more efficient treatment option for future melanoma therapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Chemokines; Drug Synergism; Gene Knockdown Techniques; Humans; Interferon Inducers; Interferon-beta; Macrophages; Melanoma; Poly I-C; RNA, Small Interfering; Signal Transduction; Skin Neoplasms; Toll-Like Receptor 3; Transcriptome; Tretinoin

Although the concept of cancer stem cells (CSCs) is well-accepted for many tumors, the existence of such cells in human melanoma has been the subject of debate. In this study, we demonstrate the existence of human melanoma cells that fulfill the criteria for CSCs (self-renewal and differentiation) by serially xenotransplanting cells into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. These cells possess high aldehyde dehydrogenase (ALDH) activity with ALDH1A1 and ALDH1A3 being the predominant ALDH isozymes. ALDH-positive melanoma cells are more tumorigenic than ALDH-negative cells in both NOD/SCID mice and NSG mice. Biological analyses of the ALDH-positive melanoma cells reveal the ALDH isozymes to be key molecules regulating the function of these cells. Silencing ALDH1A by siRNA or shRNA leads to cell cycle arrest, apoptosis, decreased cell viability in vitro, and reduced tumorigenesis in vivo. ALDH-positive melanoma cells are more resistant to chemotherapeutic agents and silencing ALDH1A by siRNA sensitizes melanoma cells to drug-induced cell death. Furthermore, we, for the first time, examined the molecular signatures of ALDH-positive CSCs from patient-derived tumor specimens. The signatures of melanoma CSCs include retinoic acid (RA)-driven target genes with RA response elements and genes associated with stem cell function. These findings implicate that ALDH isozymes are not only biomarkers of CSCs but also attractive therapeutic targets for human melanoma. Further investigation of these isozymes and genes will enhance our understanding of the molecular mechanisms governing CSCs and reveal new molecular targets for therapeutic intervention of cancer.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Aldehyde Oxidoreductases; Animals; Apoptosis; Cell Transformation, Neoplastic; Dacarbazine; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Isoenzymes; Melanoma; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Neoplastic Stem Cells; Response Elements; Retinal Dehydrogenase; RNA, Small Interfering; Skin Neoplasms; Temozolomide; Tretinoin

Topics: Antineoplastic Agents; Cell Line, Tumor; Conjunctival Neoplasms; Humans; Melanoma; Mitomycin; Nitrosourea Compounds; Organophosphorus Compounds; Tretinoin

Many melanoma cells are resistant to the anti-proliferative effect of all trans retinoic acid (ATRA). Retinoic Acid Receptor-beta2 (RAR-beta2) mediates the ATRA growth inhibition. We found a correlation between the anti-proliferative activity of ATRA and expression of RAR-beta2. There was not a strict correlation between DNA methylation of RAR-beta gene and its expression. There was no difference in global and RARbeta specific nucleosome repeat length (NRL) in melanoma and melanocytes or between control and ATRA treated cells. Pan-acetylation of H3 and H4 within the RAR-beta gene promoter was higher in cells expressing RAR-beta2. All trans retinoic acid treatment of responsive cells did not change pan-acetylation of H3/H4, but addition of ATRA to non-responsive cells increased H4 pan-acetylation. Phytochemicals or the histone deacetylase inhibitor Trichostatin A did not restore expression of RAR-beta2. Treatment of WM1366 melanoma cells with 5-aza 2'-deoxycytidine reactivated RAR-beta2 gene expression and restored the ability of ATRA to further induce the expression of this gene. Therefore, promoter methylation is responsible for silencing of RAR-beta2 in some melanoma cells and pan-acetylation of H3 likely plays a permissive role in expression of RAR-beta2.

Topics: Acetylation; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; Disease Progression; DNA Methylation; Gene Expression Regulation, Neoplastic; Gene Silencing; Histones; Humans; Melanocytes; Melanoma; Nucleosomes; Promoter Regions, Genetic; Protein Processing, Post-Translational; Receptors, Retinoic Acid; RNA, Messenger; Skin Neoplasms; Tretinoin

Lipocalin-type prostaglandin D synthase (L-PGDS) catalyses the formation of prostaglandin D(2) (PGD(2)) and also functions as a transporter for lipophilic ligands, including all-trans retinoic acid (RA). Here, we show that human epidermal melanocytes produce and secrete L-PGDS and PGD(2) in culture medium, whereas L-PGDS is not expressed in human melanoma cell lines, HMV-II, SK-MEL-28, 624 mel and G361. Treatment with RA (1 or 10 microM) for 4 days decreased the proliferation of melanocytes (30% decrease), but not melanoma cells. We therefore isolated L-PGDS-expressing cell lines from 624 mel cells. Treatment with RA decreased the proliferation of L-PGDS-expressing cells by 20%, but not mock-transfected cell lines lacking L-PGDS expression. RA induced expression of a cyclin-dependent kinase inhibitor p21(Cip1) in L-PGDS-expressing cells, but not mock-transfected cells. Moreover, RA increased the transient expression of a reporter gene carrying the RA-responsive elements in L-PGDS-expressing cell lines (at least 5-fold activation), compared to the 2-fold activation in mock-transfected cell lines, suggesting that L-PGDS may increase the sensitivity to RA. Lastly, the knockdown of L-PGDS expression by RNA interference was associated with the restoration of the RA-mediated decrease in proliferation of human and mouse melanocytes. In conclusion, L-PGDS may fine-tune the RA signalling in melanocytes.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Humans; Intramolecular Oxidoreductases; Lipocalins; Melanocytes; Melanoma; Mice; RNA Interference; Signal Transduction; Tretinoin

To investigate the effect of all-trans retinoic acid on the proliferation of and Fas protein expression in malignant melanoma A375 cells in vitro.. Malignant melanoma A375 cells cultured under different doses of all-trans retinoic acid (ATRA) and solvent controls. The effect of ATRA on cell proliferation was observed. The growth rate of the cells was detected by MTT assay. The Fas protein expression was detected by Western blotting.. ATRA inhibited the proliferation of A375 cells under 1-100 micromol/L of concentrations. The peak effect occurred after 72 hours of ATRA treatment, ATRA inhibited the growth of A375 cells in a dose and time-dependent manner. The level of Fas protein was up-regulated after exposure to 10 micromol/L of ATRA.. ATRA inhibits A375 cell proliferation perhaps through Fas death receptor pathway which induces A375 cell apoptosis.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; fas Receptor; Humans; Melanoma; Tretinoin; Up-Regulation

Treatments for primary and metastatic melanomas are rarely effective. Even therapeutics such as retinoic acid (RA) that are successfully used to treat several other forms of cancer are ineffective. Recent evidence indicates that the antiproliferative effects of RA are mediated by the transcription factor SOX9 in human cancer cell lines. As we have previously shown that SOX9 is expressed in normal melanocytes, here we investigated SOX9 expression and function in human melanomas. Although SOX9 was expressed in normal human skin, it was increasingly downregulated as melanocytes progressed to the premalignant and then the malignant and metastatic states. Overexpression of SOX9 in both human and mouse melanoma cell lines induced cell cycle arrest by increasing p21 transcription and restored sensitivity to RA by downregulating expression of PRAME, a melanoma antigen. Furthermore, SOX9 overexpression in melanoma cell lines inhibited tumorigenicity both in mice and in a human ex vivo model of melanoma. Treatment of melanoma cell lines with PGD2 increased SOX9 expression and restored sensitivity to RA. Thus, combined treatment with PGD2 and RA substantially decreased tumor growth in human ex vivo and mouse in vivo models of melanoma. The results of our experiments targeting SOX9 provide insight into the pathophysiology of melanoma. Further, the effects of SOX9 on melanoma cell proliferation and RA sensitivity suggest the encouraging possibility of a noncytotoxic approach to the treatment of melanoma.

Topics: Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Humans; Melanoma; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Nude; Microphthalmia-Associated Transcription Factor; Nevus; Prostaglandin D2; Skin Neoplasms; SOX9 Transcription Factor; Tretinoin; Up-Regulation

We assessed the effects of ATRA and retinol on melanogenesis in murine B16 melanoma cells. In the present study, ATRA and retinol inhibited melanin synthesis in melanoma cells stimulated by alpha-melanocyte stimulating hormone (alpha-MSH) or 3-isobutyl-1-methylxanthine (IBMX). To elucidate the target points of ATRA and retinol on melanogenesis, we performed western blotting for melanogenic proteins, such as tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. ATRA inhibited the expression of tyrosinase and TRP-1, and retinol inhibited the expression of tyrosinase, in a dose-dependent manner. Neither ATRA nor retinol inhibited TRP-2 expression. There were no differences in melanogenic protein expression between the two stimulants tested, alpha-MSH and IBMX. Therefore, the depigmenting effect of ATRA and retinol might be due to inhibition of the signaling pathway between cAMP and tyrosinase transcription bound to tyrosinase expression. These results indicate that ATRA and retinol are candidate anti-melanogenic agents that they might be effective in hyperpigmentation disorders.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; Melanins; Melanoma; Mice; Monophenol Monooxygenase; Pigmentation; Tretinoin; Vitamin A

The incidence of malignant melanoma has significantly increased over the last decade. Some of these malignancies are susceptible to the growth inhibitory and pro-differentiating effects of all-trans-retinoic acid (RA). The molecular changes responsible for the biological activity of RA in melanoma are not well understood.. In an analysis of sequential global gene expression changes during a 4-48 h RA treatment of B16 mouse melanoma cells, we found that RA increased the expression of 757 genes and decreased the expression of 737 genes. We also compared the gene expression profile (no RA treatment) between non-malignant melan-a mouse melanocytes and B16 melanoma cells. Using the same statistical test, we found 1495 genes whose expression was significantly higher in melan-a than in B16 cells and 2054 genes whose expression was significantly lower in melan-a than in B16 cells. By intersecting these two gene sets, we discovered a common set of 233 genes whose RNA levels were significantly different between B16 and melan-a cells and whose expression was altered by RA treatment. Within this set, RA treatment altered the expression of 203 (87%) genes toward the melan-a expression level. In addition, hierarchical clustering showed that after 48 h of RA treatment expression of the 203 genes was more closely related to the melan-a gene set than any other RA treatment time point. Functional analysis of the 203 gene set indicated that RA decreased expression of mRNAs that encode proteins involved in cell division/cell cycle, DNA replication, recombination and repair, and transcription regulation. Conversely, it stimulated genes involved in cell-cell signaling, cell adhesion and cell differentiation/embryonic development. Pathway analysis of the 203 gene set revealed four major hubs of connectivity: CDC2, CHEK1, CDC45L and MCM6.. Our analysis of common genes in the 48 h RA-treatment of B16 melanoma cells and untreated B16 vs. melan-a data set show that RA "normalized" the expression of genes involved in energy metabolism, DNA replication, DNA repair and differentiation. These results are compatible with the known growth inhibitory and pro-differentiating effects of RA. Pathway analysis suggests that CDC2, CHEK1, CDC45L and MCM6 are key players in mediating the biological activity of RA in B16 melanoma cells.

Topics: Animals; Cell Differentiation; Cell Line, Tumor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Melanocytes; Melanoma; Mice; Mice, Inbred C57BL; Oligonucleotide Array Sequence Analysis; RNA, Neoplasm; Time Factors; Tretinoin

Melanoma represents one of the most rapidly metastasizing, hence deadly tumors due to its high proliferation rate and invasiveness, characteristics of undifferentiated embryonic tissues. Given the absence of effective therapy for metastatic melanoma, understanding more fully the molecular mechanisms underlying melanocyte differentiation may provide opportunities for novel therapeutic intervention. Here we show that in mouse melanoma S91 cells activation of the peroxisome proliferator activated receptor (PPAR) gamma induces events resembling differentiation, such as growth arrest accompanied by apoptosis, spindle morphology and enhanced tyrosinase expression. These events are preceded by an initial transient increase in expression from the Microphthalmia-associated transcription factor gene, (MITF) promoter, whereas exposure to a PPAR gamma ligand- ciglitazone that exceeds 8 h, causes a gradual decrease of MITF, until by 48 h MITF expression is substantially reduced. Beta-catenin, an MITF transcriptional activator, shows a similar pattern of decline during ciglitazone treatment, consistent with previous reports that activated PPAR gamma inhibits the Wnt/beta-catenin pathway through induction of beta-catenin proteasomal degradation. We suggest that the PPAR gamma-mediated beta-catenin down-regulation is likely to be responsible for changes in MITF levels. The data suggest that PPAR gamma, besides its well-established role in mesenchymal cell differentiation towards adipocytes, might regulate differentiation in the melanocytic lineage.

Topics: Alitretinoin; Animals; Antigens, Differentiation; Antineoplastic Agents; beta Catenin; Blotting, Western; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Shape; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Melanocytes; Melanoma; Mice; Microphthalmia-Associated Transcription Factor; Monophenol Monooxygenase; Phenotype; PPAR gamma; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Thiazolidinediones; Tretinoin; Up-Regulation

The current therapy of uveal melanoma (UM) metastases remains inefficient, which warrants the development of new treatment modalities. For the first time we investigated the effects of retinoic acid (RA) on a panel of UM cell lines and found that RA induces morphological changes compatible with differentiation, suppresses proliferation and causes apoptosis in these cells. RA treatment resulted in an increase of p21, p27 and p53 protein levels and G1 arrest in UM cells, which correlated with significant down-modulation of surface Her2/neu proto-oncogene expression. In addition, RA-treated UM cells exhibited increased sensitivity to both MHC class I-restricted killing by cytotoxic T lymphocytes and NK cell-mediated lysis that were accompanied by more efficient conjugate formation between UM cells and killer lymphocytes. Taken together, our results implicate UM as a new target for treatment with retinoids and suggest that retinoids and T- or NK-cell based immunotherapy can have mutually enhancing effects in UM patients.

Topics: Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cytotoxicity, Immunologic; Flow Cytometry; Humans; Killer Cells, Natural; Melanoma; Proliferating Cell Nuclear Antigen; Proto-Oncogene Mas; Proto-Oncogene Proteins p21(ras); T-Lymphocytes; Tretinoin; Tumor Suppressor Protein p53; Uveal Neoplasms

Incidence of melanoma continues to rise, and a better understanding of its genetics will be critical to improve diagnosis and develop new treatments. Here, we search for novel melanoma-specific genes that may serve as biomarkers and therapeutic targets by using an in vitro genetic screen. One identified cDNA encoded TROY, a member of the tumor necrosis factor receptor superfamily (TNFRSF). TROY is widely expressed during embryogenesis, but in adults expression is restricted to hair follicles and brain. However, TROY had never been associated with melanoma, and it was selected for further study. First we show that expression in melanoma is specific by semiquantitative RT-PCR analysis of a large panel of established tumor cell lines. Next, specificity of expression was evaluated by immunohistochemistry analysis of primary cell cultures and patient tissues. TROY is expressed in 2/2 primary melanoma cells and 45/45 melanoma tissue samples (p < 0.0001). With the exception of sebaceous glands, TROY is not expressed in normal skin biopsies (p < 0.0001) or primary skin cell cultures that contain keratinocytes and epidermal melanocytes, nor is it expressed in other skin tumor cells (p < 0.0001). Finally, we show that TROY regulates melanoma growth, because replication of melanoma cells with reduced TROY levels through treatment with short-interfering RNA was significantly decreased relative to control cells (p < 0.004). In summary, TROY is the first TNFRSF member that is a biomarker for melanoma. TROY also presents a potentially novel cell surface signaling target for inhibitors, cell and/or antibody-based immunotherapies.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Melanocytes; Melanoma; Receptors, Tumor Necrosis Factor; RNA, Messenger; Skin; Skin Neoplasms; TNF Receptor-Associated Factor 6; Tretinoin

Natural killer (NK) cells play a role in the innate and adaptive antitumor immune responses. The activity of NK cells is regulated by functionally opposing, activating and inhibitory receptors whose balance ultimately determines whether target cells will be susceptible to NK cell mediated lysis. As melanoma is an immunogenic tumor, the effect of immunomodulating agents is consistently investigated. In this study in 79 metastatic melanoma (MM) patients and 52 controls NK activity, expression of activating NKG2D and CD161 receptors and KIR receptors, CD158a and CD158b, on freshly isolated PBL and NK cells were evaluated. Native NK cell activity of melanoma patients in clinical stage I-III and MM patients was determined against NK sensitive K562, NK resistant Daudi, human melanoma FemX, HeLa and HL 60 target tumor cell lines. In addition, predictive pretherapy immunomodulating effect after 18 h in vitro treatments of PBL of MM patients with rh IL-2, IFN-alpha (IFN), 13-cis retinoic acid (RA) and combination IFN-alpha and RA was evaluated with respect to NK cell lyses against K562 and FemX cell lines. In this study we show for the first time that low expression of CD161 and activating NKG2D receptors, without increased expression of KIR receptors CD158a and CD158b, as well as a decrease in the cytotoxic, CD16(bright) NK cell subset, is associated with a significant impairment in NK cell activity in MM patients. Furthermore, the predictive pretherapy finding that IL-2, IFN, IFN and RA, unlike RA alone, can enhance NK cell activity of MM patients against FemX melanoma tumor cell line can be of help in the design and development of therapeutic regimens, considering that it has recently been shown that low-dose combination of different immunomodulators represents the most promising approach in the therapy of MM.

Topics: Adult; Aged; Antigens, Surface; Antineoplastic Agents; Cell Line, Tumor; Cytotoxicity, Immunologic; Female; Flow Cytometry; History, 17th Century; Humans; Interferon-alpha; Interleukin-2; Killer Cells, Natural; Lectins, C-Type; Lymphocytes; Male; Melanoma; Neoplasm Metastasis; NK Cell Lectin-Like Receptor Subfamily B; NK Cell Lectin-Like Receptor Subfamily K; Receptors, Immunologic; Receptors, KIR; Receptors, KIR2DL1; Receptors, KIR2DL3; Receptors, Natural Killer Cell; Recombinant Proteins; Skin Neoplasms; Tretinoin

TR3 was originally recognized for its role in the regulation of cell survival and differentiation, however, it was recently found to be a potent pro-apoptotic protein. In order to characterize the role of TR3 in melanoma cell apoptosis, we studied expression of TR3 in melanoma cell lines and tissues, its subcellular distribution and function during apoptosis using various expression and RNA interference vectors. We found that TR3 was constitutively expressed in both cultured melanoma cells and melanoma tissues. TR3 expression was significantly decreased in advanced melanomas comparing to benign nevi. Over-expression of wild type TR3 or mutant TR3 lacking the DNA binding domain resulted in massive apoptosis in melanoma cells, whereas stable knockdown of TR3 using RNA interference resulted in melanoma cell resistance to apoptosis induced by chemotherapeutic agents ATRA and cisplatin. We further demonstrated that apoptosis in melanoma cells was mediated, at least partially, through TR3 mitochondrial translocation but not alteration in TR3 expression levels. Our results suggest that TR3 is an important apoptosis inducing factor in melanoma cells. Decreased expression of TR3 in metastatic melanoma cells may contribute to their reduced apoptotic potential and increased resistance to chemotherapy.

Topics: Apoptosis; Cell Line, Tumor; Cisplatin; Cytochromes c; DNA-Binding Proteins; Humans; Melanoma; Mitochondria; Nuclear Receptor Subfamily 4, Group A, Member 1; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Skin Neoplasms; Transcription Factors; Tretinoin

The few chemoimmunotherapy trials that together with dacarbazine (DTIC) and interferon-alpha 2a (IFNalpha), include retinoic acid (RA), did not include detailed immunological evaluation of functional and phenotypic natural killer (NK) cell characteristics, and have shown contradictory clinical results.. Malignant melanoma (MM) patients undergoing phase II-randomized chemoimmunotherapy trials were treated with DTIC, IFNalpha (Hoffmann-La Roche) (group A, n = 31), and with DTIC, IFNalpha and 13-cis-RA (Isotretinoin, Hoffmann-La Roche, Basel, Switzerland) (group B, n = 29). Patients and 42 healthy controls were evaluated by FACS flow analyses for CD3/CD56/CD69 positive cells, NK cytotoxicity in fresh peripheral blood lymphocytes (PBL) and for interferon regulatory factor-1 mRNA expression by reverse transcriptase polymerase chain reaction in treated PBL.. The addition of RA to a DTIC-IFN regime did not bring any therapeutical benefit in terms of response or survival. Immunological follow-up on days 1, 6 and 27 of each therapy cycle shows a significant increase in NK cell activity in both groups, only on day 6 of the first cycle, while CD69+CD56+ expression increased significantly on day 6 of each therapy cycle, in both groups. Evaluation of the dynamics of expression of IRF-1 of in vitro treated PBL, shows its strong and prompt up-regulation by IFNalpha and synergistic effect of IFNalpha and RA combination.. The dynamics of the increase in CD69 early activation antigen expression on CD56+ NK cells is systematic and serial with the increase being significantly higher on day six of the first cycle in group B patients with clinical response, compared to those without, indicating possible predictive value of CD69 expression for clinical response to chemoimmunotherapy.

Topics: Adult; Aged; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Antineoplastic Combined Chemotherapy Protocols; Case-Control Studies; CD56 Antigen; Dacarbazine; Female; Humans; Immunotherapy; Interferon alpha-2; Interferon Regulatory Factor-1; Interferon-alpha; Killer Cells, Natural; Lectins, C-Type; Male; Melanoma; Middle Aged; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms; Tretinoin

All-trans-retinoic acid (atRA) exerts its effects via apoptosis and cell cycle re-distribution. However, the mechanisms behind the effects have not been fully understood. In this study, we used a model system of matched primary and metastatic melanoma cells to investigate whether expression of Id1 and p16 proteins were involved in atRA-induced apoptosis and cell cycle re-distribution. Melanoma cells were exposed to 0.1 or 10 microM atRA for 1-96 h. Apoptosis and cell cycle were measured by flow cytometry. Expression of Id1 and p16 proteins was examined by Western blotting and immunocytochemistry. After exposure to atRA we found a marked increase in apoptosis and cell cycle re-distribution in both primary and metastatic melanoma cells. Expression level of Id1 protein was decreased and the p16 was increased in a dose- and time-dependent (P<0.05) manner after treatment with atRA. Alterations of these proteins were more pronounced in the primary melanoma cells than the matched metastases (P<0.05). These data suggested that the alterations of Id1 and/or p16 proteins were involved in atRA-induced apoptosis and cell cycle re-distribution in melanoma. These expression profiles of Id1 and p16 proteins may provide molecular evidence for better chemotherapy primarily for early stages of melanoma.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p16; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression Profiling; Humans; Immunohistochemistry; In Vitro Techniques; Inhibitor of Differentiation Protein 1; Melanoma; Neoplasm Proteins; Repressor Proteins; Transcription Factors; Tretinoin

To investigate the effects of all-trans retinoic acid (ATRA), acitretin and tazarotene on apoptosis and Bax/Bcl-2 protein expressions of human melanoma A375 cells.. The effects of retinoids on apoptosis and Bax/Bcl-2 protein expressions of A375 cells in vitro were examined. Apoptosis analysis with double staining with annexin V-FITC and PI was performed using flow cytometer. SABC immunocytochemistry was employed for detection of Bax/Bcl-2 protein expressions.. At the concentration of 1 x 10(-5) mol/L, ATRA, acitretin and tazarotene all induced apoptosis of A375 cells with apoptosis ratio of 5.03% (P<0.05), 13.42% (P<0.05) and 2.88% (P>0.05), respectively, and acitretin induced more significant apoptosis than the other two agents (P<0.05). In addition, all the three agents significantly increased the number of cells positive for Bax expression and decreased the number of cells expressing Bcl-2 (P<0.05), among which acitretin had the strongest effects.. ATRA, acitretin and tazarotene mediate apoptosis of A375 cells possibly through, at least partially, the mitochondrial pathway. Acitretin may be utilized as a valuable alternative for treating melanoma.

Topics: Acitretin; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Humans; Melanoma; Nicotinic Acids; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms; Tretinoin; Tumor Cells, Cultured

Topics: Apoptosis; Caspase 3; Caspases; Cell Line, Tumor; Cell Proliferation; Humans; Isotretinoin; Melanoma; Receptors, Retinoic Acid; Retinoid X Receptors; Tretinoin

Retinoic acid (RA) induces proliferation arrest, differentiation, and apoptosis, and defects in retinoic acid receptor (RAR) signaling have been implicated in cancer. The human tumor antigen PRAME is overexpressed in a variety of cancers, but its function has remained unclear. We identify here PRAME as a dominant repressor of RAR signaling. PRAME binds to RAR in the presence of RA, preventing ligand-induced receptor activation and target gene transcription through recruitment of Polycomb proteins. PRAME is present at RAR target promoters and inhibits RA-induced differentiation, growth arrest, and apoptosis. Conversely, knockdown of PRAME expression by RNA interference in RA-resistant human melanoma restores RAR signaling and reinstates sensitivity to the antiproliferative effects of RA in vitro and in vivo. Our data suggest that overexpression of PRAME frequently observed in human cancers confers growth or survival advantages by antagonizing RAR signaling.

Topics: Animals; Antigens, Neoplasm; Apoptosis; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Enhancer of Zeste Homolog 2 Protein; Humans; Ligands; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Polycomb Repressive Complex 2; Protein Binding; Receptors, Retinoic Acid; Repressor Proteins; RNA Interference; Sensitivity and Specificity; Signal Transduction; Transcription Factors; Transplantation, Heterologous; Tretinoin

Melanin is a dark pigment protecting the skin against UV radiation in some organisms. Studies on invasion and metastasis using retinoic acid as inhibitor agent are well known, but its role in melanin production (melanogenesis), especially at ultrastructural level and using morphometry were not well studied. In the present study, we analyzed the effects of retinoic acid on the melanosomes in B16F10 melanoma cells. These organelles were identified and quantified using routine electron microscopy and the specific HMB45 antibody. Other approaches such as immunofluorescence, and flow cytometry were also used. Our results indicated that retinoic acid increased the melanogenesis process in B16F10 melanoma cells. Furthermore, this work also provided evidence that this substance interferes at the subcellular level altering the numerical density of melanosomes, as well as the relative volume of the nucleus and nucleolus. In addition, the cells displayed altered morphology and an increase in the percentage of the relative volume of melanosomes, mainly the stages II-III and IV, leading to melanin formation. Furthermore, a decrease in the cells number after retinoic acid treatment was also observed.

Topics: Animals; Antineoplastic Agents; Cell Division; Cell Line, Tumor; Cell Nucleus; Image Cytometry; Melanins; Melanoma; Melanosomes; Mice; Microscopy, Confocal; Microscopy, Electron; Tretinoin

We have previously shown that all-trans-retinoic acid (atRA) induces apoptosis in melanoma cells and primary melanoma cells are more sensitive to the exposure of atRA than the matched metastases. However, mechanisms behind the atRA-induced apoptosis have not been studied. In this study, we used a similar cell culture model system of matched primary and metastatic melanoma cells from the same patient to investigate whether p53 and bcl-2 family proteins were involved in atRA-induced apoptosis. The primary and metastatic melanoma cells were exposed to 0.1 and 10 micro M atRA in serum-free RPMI 1640 cell culture medium in the dark for up to 96 h. The protein expression of p53, p21, bax and bcl-2 were examined by Western blotting and immunocytochemistry. Expression of p53, p21 and bax was increased, and bcl-2 was decreased in melanoma cells after exposure to atRA at different concentrations for various periods of time. The changes of p53, p21, bax, and bcl-2 protein levels were dose- and time-dependent. The primary melanoma cells were more sensitive to the atRA treatments than cells from matched metastatic melanoma. These data indicate that p53, p21, bax and bcl-2 proteins were involved in atRA-induced apoptosis in melanoma cells. Modification of these protein levels in the tumour cells might be beneficial for early treatment of melanoma.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Cycle Proteins; Cell Line; Cyclin-Dependent Kinase Inhibitor p21; Humans; Melanoma; Neoplasm Metastasis; Neoplasm Proteins; Proto-Oncogene Proteins c-bcl-2; Tretinoin; Tumor Suppressor Protein p53

We investigated whether p27 and mitogen-activated protein kinase (MAPK) proteins were involved in all-trans retinoic acid (atRA)-induced apoptosis and cell cycle arrest. Matched primary and metastatic melanoma cells were exposed to atRA. Apoptosis and cell cycle were detected by flow cytometry. Expression of p27, Ras, B-raf, Mek and Erk proteins was examined. Results showed that atRA induced apoptosis and cell cycle arrest in both primary and metastatic melanoma cells. The primary melanoma cells were more vulnerable than their matched metastatic cells. Expression of p27 was increased, while MAPK proteins were decreased, this response was dose- and time-dependent. Alterations of these proteins were more pronounced in primary melanoma cells than in the matched metastases. These data indicate that up-regulation of p27 and down-regulation of MAPK proteins were involved in atRA-induced apoptosis and cell cycle arrest in melanoma.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p27; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genes, Tumor Suppressor; Humans; Melanoma; Mitogen-Activated Protein Kinases; Neoplasm Metastasis; Skin Neoplasms; Tretinoin; Tumor Suppressor Proteins

Retinoic acid (RA) inhibits growth and induces differentiation of B16 mouse melanoma cells. These effects are accompanied by a large increase in PKCalpha mRNA and protein levels and surprisingly an increase in activating protein-1 (AP-1) transcriptional activity. To further investigate the RA-induced AP-1 activity we established clones of B16 cells stably expressing an AP-1-luciferase reporter gene. Treatment of these clones with phorbol dibutyrate increased AP-1 activity which peaked at 2-4 h and returned to baseline level by 24 h. In contrast, RA treatment resulted in a slow increase in AP-1 activity that reached a maximum level at 48 h and was maintained for the duration of the treatment. We tested the importance of the RA-induced AP-1 activity by establishing clones which stably express a dominant negative fos gene (A-fos) and have greatly diminished AP-1 activity. Growth rates of untreated A-fos expressing cells were similar to wt B16 and clones not expressing A-fos. However, clones expressing the dominant-negative fos had a markedly decreased sensitivity to RA-induced inhibition of anchorage-dependent and -independent growth. Treatment of wt B16 cells for 48 h with RA increased melanin production by two to fourfold, but this effect was completely lost in the A-fos clones. The ability of RA to induce RARbeta and PKCalpha expression was retained in A-fos clones, suggesting that A-fos was not interfering with RAR transcription activation functions. We tested whether the RA-induced AP-1 activity might be mediated by the ERK1/2 MAPK pathway. Inhibition of ERK1/2 phosphorylation stimulated AP-1 activity, which was not additive to that induced by RA. This finding raises the possibility that this MAPK pathway may be a target of retinoid action. Our observations suggest that AP-1 transcriptional activity induced by RA likely plays an important role in the biological changes mediated by this retinoid in B16 melanoma cells.

Topics: Animals; Bacterial Proteins; Cell Differentiation; Cell Division; Genes, Dominant; Glutathione Transferase; Isoenzymes; Melanins; Melanoma; Mice; Phorbol 12,13-Dibutyrate; Protein Kinase C; Protein Kinase C-alpha; Receptors, Retinoic Acid; Signal Transduction; Transcription Factor AP-1; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

All-trans retinoic acid (atRA) has been suggested to exert its cytotoxicity via apoptosis but the mechanisms behind the damage effects have not been fully understood. In this study, we investigated the cytotoxic effects of atRA in eleven primary and matched metastatic cutaneous melanoma cell lines. All the primary and metastatic melanoma cell lines examined expressed the retinoic acid receptors. The cultured melanoma cells treated with atRA showed dysfunction of mitochondria and altered cell cycle distribution, inhibited cell proliferation and apoptosis. The cytotoxic effects of atRA were dose- and time-dependent. The dysfunction of mitochondria and induction of apoptosis were more pronounced in the primary tumor cells than in the metastatic cell lines from the same patients. The data indicate that the cytotoxic effect of atRA was mediated through dysfunction of mitochondria, alterations in cell cycle and induction of apoptosis. Melanoma in early stage may have better response to atRA adjuvant therapy than the melanoma in late stage, suggesting the early utility of atRA in melanoma chemotherapy.

Topics: Apoptosis; Cell Cycle; Flow Cytometry; Melanoma; Mitochondria; Neoplasm Metastasis; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) induces growth-arrest of many tumor cell lines but it is an ineffective therapeutic against melanoma. We investigated whether the histone deacetylase (HDAC)-inhibitor sodium butyrate (BUT) can restore or potentiate the RA-response of RA-resistant human A375, and RA-responsive S91 murine melanoma cells. BUT induced expression of RARbeta and p21(waf1/cip1) mRNA in A375 cells but in S91 cells only p21(waf1/cip1) was induced. RA and BUT synergistically activated transcription of an RA-dependent reporter gene in S91, but not A375 cells. BUT increased histone H4-acetylation in both cell types. RA potentiated BUT-mediated inhibition of S91 cell proliferation, whereas A375 cells remained largely resistant to both compounds. HDAC-inhibitors may enhance the activity of RA on RA-responsive melanoma cells.

Topics: Animals; Antineoplastic Agents; Blotting, Northern; Butyrates; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Luciferases; Melanoma; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Response Elements; RNA, Messenger; Transfection; Tretinoin; Tumor Cells, Cultured

Tyrosinase-related protein 2 (TRP-2), also known as DOPAchrome tautomerase, is an enzyme in melanin biosynthesis and may play an important role in detoxification of a metabolite derived from DOPA. TRP-2 is expressed in melanocytes of neural crest origin and retinal pigment epithelium (RPE), derived from the optic cup. TRP-2 has been established as an early differentiation marker for melanoblasts and RPE. It is therefore of significance to study the regulation of TRP-2/DOPAchrome tautomerase expression. Here we show that TRP-2 mRNA is expressed in Y79 human retinoblastoma cell line, derived from a primitive multipotential retinal cell. Retinoblastoma is the common primary intraocular tumor of childhood. Basal expression levels in Y79 retinoblastoma cells of TRP-2 mRNA and protein are comparable to those in melanoma cells, whereas mRNA for tyrosinase, the rate-limiting enzyme in melanogenesis, is undetectable in retinoblastoma cells. Transient transfection assays showed that the TRP-2 gene promoter efficiently directs the reporter gene expression in retinoblastoma cells as it does in melanoma cells. Moreover, the expression of TRP-2 mRNA was induced by retinoic acid in retinoblastoma cells but not noticeably affected by forskolin, a cAMP-elevating reagent, whereas in melanoma cells its expression was induced by forskolin but not by retinoic acid. These results suggest a difference in the regulation of TRP-2 expression between retinoblastoma and melanoma cells. Moreover, TRP-2 mRNA is expressed in the excised retinoblastoma specimens, as assessed by RT-PCR. The present study shows unexpected features of TRP-2 and may enhance our understanding of the pathophysiology of retinoblastoma.

Topics: Colforsin; Gene Expression; Genes, Reporter; Humans; Melanoma; Monophenol Monooxygenase; Retinal Neoplasms; Retinoblastoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Two new canine melanoma cell lines (CMM1 and CMM2) were established from the patients with oral malignant melanomas. Histopathological type of both CMM1 and CMM2 was a mixed cell type consisted of spindle-shaped cells, polygonal cells, and oval cells. Doubling time of CMMI and CMM2 were 18.4 +/- 1.96 hr and 21.0 +/- 0.73 hr, respectively. The effect of two kinds of retinoids (all-trans retinoic acid and 9-cis retinoic acid) on the proliferation of these cells were examined by morphological changes, proliferation assay and apoptosis assay. However, the retinoids did not suppress growth rate of these cells. This result suggests that retinoids used in this study did not induce differentiation, apoptosis, and growth inhibition of the canine melanoma cell lines.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Apoptosis; Cell Division; Dog Diseases; Dogs; Melanoma; Mouth Neoplasms; Retinoids; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) slows growth and induces differentiation of tumor cells through activation of RA receptors (RARs). However, melanoma cell lines display highly variable responsiveness to RA, which is a poorly understood phenomenon. By using Northern and Western blot analyses, we show that RA-resistant A375 and RA-responsive S91 melanoma cells express comparable levels of major components of RAR-signaling pathways. However, A375 cells have substantially higher intracellular reactive oxygen species (ROS) levels than S91 cells. Lowering ROS levels in A375 cells through hypoxic culture conditions restores RAR-dependent trans-activity, which could be further enhanced by addition of the antioxidant N-acetyl-cysteine. Hypoxia also enhances RAR activity in the moderately RA-responsive C32 cells, which have intermediate ROS levels. Conversely, increasing oxidative stress in highly RA-responsive S91 and B16 cells, which have low ROS levels, by treatment with H(2)O(2) impairs RAR activity. Consistent with these observations, RA more potently inhibited the proliferation of hypoxic A375 cells than that of normoxic cells. Oxidative states diminish, whereas reducing conditions enhance, DNA binding of retinoid X receptor/RAR heterodimers in vitro, providing a molecular basis for the observed inverse correlation between RAR activity and ROS levels. The redox state of melanoma cells provides a novel, epigenetic control mechanism of RAR activity and RA resistance.

Topics: Acetylcysteine; Antioxidants; Blotting, Northern; Blotting, Western; Cell Hypoxia; Dimerization; DNA; Drug Resistance, Neoplasm; Gene Expression; Hydrogen Peroxide; Luciferases; Melanoma; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Receptors, Retinoic Acid; Recombinant Fusion Proteins; Retinoid X Receptors; Signal Transduction; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

Studies into the effects of topical retinoic acid on photocarcinogenesis have yielded ambiguous findings. This may be due to different Experimental protocols and ultraviolet spectra. Retinoic acid is commonly used for a range of dermatologic conditions, and therefore it is important to resolve whether it affects skin tumor formation. To address this issue we used a protocol to mimic as closely as possible human use of retinoic acid. Two mouse strains were used: Skh:HR-1 (albino) and Skh:HR-2 (lightly pigmented). The pigmented mice more closely resemble Caucasian skin as they develop a light tan in response to ultraviolet radiation. This tan is greatly augmented by retinoic acid. As these are congenic mice, any differences can be attributed to the development of a tan. Mice were exposed to solar-simulated ultraviolet radiation, followed by treatment with 0.05% retinoic acid. This modeled exposure to sunlight during the day followed by retinoic acid treatment and a night-time period in the absence of sunlight. As it is recommended that ultraviolet exposure is minimized when using topical retinoic acid, the mice were only exposed to one-third of minimal edemal dose of ultraviolet radiation per day. This retinoic acid protocol augmented photocarcinogenesis. Retinoic acid decreased the latency period, reduced the probability that a mouse would survive without a tumor, and increased the number of tumors per mouse. All tumors induced were squamous cell carcinomas, and the skin between the tumors on mice treated with retinoic acid was found to contain carcinoma in situ upon histologic diagnosis. The light tan of the solvent-treated pigmented mice did not provide any protection, whereas the dark tan, which developed in Skh:HR-2 mice in response to retinoic acid, reduced photocarcinogenesis but did not overcome the augmenting effect of retinoic acid. Thus, using this experimental design, topical retinoic acid augmented photocarcinogenesis, and the ability to develop a dark but not light tan provided some, but limited, protection.

Topics: Administration, Topical; Animals; Carcinoma, Squamous Cell; Female; Melanins; Melanoma; Mice; Pigmentation; Skin Neoplasms; Sunlight; Tretinoin; Ultraviolet Rays

The intriguing problem of metastasis requires the spreading of metastatic cells through the basement membrane barrier. The interaction of the basement membrane with the metastatic cell is a cell surface activity involving the function of integrin receptors. Integrins are a group of alpha,beta heterodimeric proteins responsible for transducing intracellular signals on binding to the extracellular matrix proteins present in the basement membrane. To understand the role of integrin receptors in tumor metastasis, the cell surface receptor functions were modulated by All Trans Retinoic Acid (ATRA) treatment in B16F10 tumor cells. Our experimental results clearly indicate that All Trans Retinoic Acid (ATRA) inhibit metastatic potential of highly metastatic B16F10 melanoma cells by 1) downregulating the cell surface integrin receptors against ECM proteins specially laminin and vitronectin and 2) by inhibiting the 72 kd collagenase activity.

Topics: Antineoplastic Agents; Extracellular Matrix Proteins; Humans; Integrins; Melanoma; Neoplasm Metastasis; Signal Transduction; Tretinoin; Tumor Cells, Cultured

Intercellular adhesion molecule (ICAM-1) exists as a membrane-associated form (mICAM-1) on the surface of tumour cells as well as a soluble form (sICAM-1). This study analyses the ability of all-trans retinoic acid (RA) to alter both sICAM and mICAM-1 expression in C8161 and Hs294T human melanoma cell lines and investigates the involvement of ICAM-1 in the interaction between tumour and lymphokine-activated killer (LAK) cells using the Cr-51 release assay. Our data showed that 4-day pretreatment of the tumour cells with 10(-7) M RA and 10(-6) M RA induced an increase in lysis of both cell lines and also increased mICAM-1 expression without having any effect on sICAM-1 levels. Addition of blocking ICAM-1 antibody (10 microg ml(-1)) to the C8161 cells at an effector:tumour cell ratio of 40:1 caused a 2.3-fold reduction in lysis of tumour cells and a 3-fold reduction in lysis of RA-treated cells. Blocking ICAM-1 antibody at optimum concentrations of 5 microg ml(-1) reduced lysis 1.8-fold in control Hs294T cells and 1.3-fold in RA-treated cells. Blocking the HLA-ABC complex had no effect on lysis. The more highly metastatic C8161 cells were found to secrete 4-fold greater levels of sICAM-1 than the poorly metastatic Hs294T cells and addition of sICAM-1 to the assay failed to affect lysis of either cell line but did induce a 2-fold decrease in lysis of RA-treated C8161 cells. Collectively, these data provide further evidence for ICAM-1 involvement in the tumour/LAK cell response and indicates that the RA-induced increase in mICAM-1 levels are partly responsible for the increase in susceptibility of the tumour cells. sICAM-1 appears to be unimportant in evasion of the tumour cells from LAK cell lysis, but may play a role in evasion of RA-treated C8161 cells.

Topics: Antibodies; Cell Survival; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Fluorescent Antibody Technique; Humans; Intercellular Adhesion Molecule-1; Killer Cells, Lymphokine-Activated; Melanoma; Tretinoin; Tumor Cells, Cultured

Along with degradation of type IV collagen in basement membrane, destruction of the stromal collagens, types I and III, is an essential step in the invasive/metastatic behavior of tumor cells, and it is mediated, at least in part, by interstitial collagenase 1 (matrix metalloproteinase 1 (MMP-1)). Because A2058 melanoma cells produce substantial quantities of MMP-1, we used these cells as models for studying invasion of type I collagen. With a sensitive and quantitative in vitro invasion assay, we monitored the ability of these cells to invade a matrix of type I collagen and the ability of a serine proteinase inhibitor and all-trans-retinoic acid to block invasion. Although these cells produce copious amounts of MMP-1, they do not invade collagen unless they are co-cultured with fibroblasts or with conditioned medium derived from fibroblasts. Our studies indicate that a proteolytic cascade that depends on stromal/tumor cell interactions facilitates the ability of A2058 melanoma cells to invade a matrix of type I collagen. This cascade activates latent MMP-1 and involves both serine proteinases and MMPs, particularly stromelysin 1 (MMP-3). All-trans-retinoic acid (10(-6) M) suppresses the invasion of tumor cells by several mechanisms that include suppression of MMP synthesis and an increase in levels of tissue inhibitor of metalloproteinases 1 and 2. We conclude that invasion of stromal collagen by A2058 melanoma cells is mediated by a novel host/tumor cell interaction in which a proteolytic cascade culminates in the activation of pro-MMP-1 and tumor cell invasion.

Topics: Antineoplastic Agents; Collagen; Collagenases; Enzyme Activation; Humans; Matrix Metalloproteinase 1; Melanoma; Neoplasm Invasiveness; Tretinoin; Tumor Cells, Cultured

The plasminogen activation system comprises various proteases that contribute to the invasive potential and metastatic spread of the tumour cell. Two such proteases are tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. Both these enzymes convert plasminogen into the active zymogen plasmin, which has a broad substrate specificity and is capable of degrading a wide range of extracellular matrix molecules. In this study, we examined the effect of retinoic acid (RA) on uPA and tPA secretion in the highly metastatic C8161 and the poorly metastatic Hs294T human melanoma cell lines using a specific enzyme-linked immunosorbent assay (ELISA) detection system, and correlated this production with RA receptor (RAR) expression. Over a range of dilutions, we were able to show that the highly metastatic C8161 cells secreted 0.95 ng of uPA/cell compared with 4.41 fg/cell for the Hs294T cells, whereas the Hs294T cells secreted 24.5 fg of tPA/cell compared with 4.35 fg/cell for the C8161 cells. On exposure of the cells to RA (10(-10)-10(-5) M) for 4 days, uPA secretion was increased 3.4-fold in the C8161 cell line and 1.6-fold in the Hs294T cell line using 10(-8) M RA. In addition, tPA expression was increased in both cell lines by 3.7-fold in the C8161 cells and 3.8-fold in the Hs294T cells with 10(-6) M RA treatment. Increases in PA expression by RA have been reported to involve RAR alpha and RAR beta expression. We were able to detect RAR beta and gamma expression in both cell lines, with and without RA treatment, but were unable to detect expression of RAR alpha. This suggests that another mechanism must exist to regulate the RA modulation of tPA and uPA secretion in these cell lines that does not require RAR alpha expression.

Topics: Blotting, Southern; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Gene Expression; Humans; Melanoma; Receptors, Retinoic Acid; Tissue Plasminogen Activator; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The purpose of this study was to investigate the effects of retinoid analogues with different retinoid receptor specificity on the growth of human D10 and Cloudman S91 mouse melanoma cells. We compared the growth inhibitory effects with the ability of retinoids to downregulate cell surface expression of the melanocortin receptor (MC1-R). Retinoic acid receptor (RAR)-gamma-selective retinoids exerted the most prominent growth effects, with up to 68% and 69% inhibition in D10 and S91 cells, respectively. A retinoid X receptor (RXR)-selective compound inhibited cell growth by only 14% and 23% in D10 and S91 cells, respectively. Growth inhibition by RARalpha- and RARbeta-selective compounds was below 10% in both cells. In D10 cells, MC1-R downregulation was also induced most effectively by an RARgamma-selective retinoid (84% relative to controls). RARalpha-, RARbeta-and RXR-selective agonists induced only 16-24% MC1-R downregulation in these cells. The pattern for MC1-R downregulation was completely different in S91 cells. The RXR-selective compound was the most active (85%), followed by the RARalpha-selective agonist (58%), the RARgamma-selective compound (47%), and finally by the RARbeta-selective agonist (29%). We conclude that RARgamma-selective retinoids may have potential as therapeutic agents in melanoma. Different selectivity profiles for growth inhibition and MC1-R downregulation in S91 cells suggest that these two retinoid effects are not directly dependent on each other.

Topics: Animals; Cell Division; Cyclic AMP; Down-Regulation; Humans; Melanoma; Melanoma, Experimental; Mice; Receptors, Corticotropin; Receptors, Melanocortin; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Retinoid X Receptors; Retinoids; Structure-Activity Relationship; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Invasive growth and formation of metastases involve complex interactions between tumour cells, host cells and components of the extracellular matrix. Retinoids, a group of vitamin A derivatives, modulate cell growth and differentiation and have been found to suppress tumour cell invasion in vitro and formation of metastases in vivo. The aim of our study was to investigate changes in proliferation and invasion through membrane barriers in vitro of seven human melanoma cell lines, established from human primary melanomas or metastases, in response to treatment with retinoic acid (RA). These changes were compared with the expression regulation of molecules that have been identified as targets of RA-mediated signal pathways. Invasiveness in vitro was correlated with the origin of the cell lines and was significantly higher in the lines derived from metastases. In all the cell lines proliferation and chemotaxis were inhibited by 10(-5) M RA, but the cell lines established from metastases were significantly more sensitive with respect to inhibition of invasion by RA. The specific expression patterns of MMP-1 and TIMP-2 were detected and regulated by RA in almost all cell lines, whereas expression of MMP-2 and TIMP-1 was not influenced by RA treatment. The most striking difference between the cell lines was a strong downregulation of transforming growth factor-beta (TGF-beta) expression in cell lines derived from metastases when treated with RA in contrast to cell lines from primary melanomas. These data provide evidence that RA modulates growth, chemotaxis and invasion in a broad panel of melanoma cell lines derived both from primary non-metastasized melanomas and metastases. However, distinct molecular mechanisms are involved in mediating these effects.

Topics: Antineoplastic Agents; Blotting, Northern; Cell Division; Chemotaxis; Collagen; Collagenases; Dose-Response Relationship, Drug; Drug Combinations; Enzyme-Linked Immunosorbent Assay; Gelatinases; Gene Expression Regulation; Humans; Laminin; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanoma; Metalloendopeptidases; Neoplasm Invasiveness; Neoplasm Proteins; Protease Inhibitors; Proteoglycans; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Preliminary data have shown that IL-6 may act as an autocrine growth factor to control proliferation. We further characterised the role of IL-6 in tumour growth as an autocrine/paracrine growth factor in neuroectodermal tumours. We evaluated the production and secretion of IL-6 by seven human melanoma, five neuroblastoma and one glioblastoma cell lines. Moreover, we determined their IL-6-dependent growth in serum free-medium or under minimal growth-supplement conditions: IL-6 dependent growth was observed in two non-IL-6 producing melanoma and in one neuroblastoma cell lines. In addition, expression of IL-6 mRNA and peptide was increased by retinoic acid. The data support the hypothesis that IL-6 contributes to neuroectodermal tumour growth, even though it shows a less potent effect than other reported growth factor such as IGF-II.

Topics: Antineoplastic Agents; Blotting, Northern; Cell Division; Culture Media, Serum-Free; Gene Expression Regulation, Neoplastic; Glioblastoma; Growth Substances; Humans; Interleukin-6; Melanoma; Neuroblastoma; Neuroectodermal Tumors; Radioimmunoassay; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) induces differentiation of B16 mouse melanoma cells, which is accompanied by an increase in protein kinase Calpha (PKCalpha) as well as a selective enrichment of nuclear PKCalpha. We report here that RA also increases AP-1 activity in these cells. Transient transfection of B16 cells with luciferase reporter gene constructs indicated that RA induced a concentration-dependent increase in AP-1 activity. Acute treatment (2 h) of B16 cells with phorbol dibutyrate (PDB) increased AP-1 activity by 10-fold. RA treatment did not change the expression of Jun family members; however, it decreased the expression of c-Fos. In contrast acute PDB treatment induced c-Fos expression, while having little effect on c-Jun. Five DNA-protein complexes were formed with nuclear extracts from B16 cells and an oligonucleotide containing an AP-1 consensus sequence. Several complexes were decreased in cells treated with RA. Conversely, certain complexes were increased in cells acutely treated with PDB. The slowest migrating complexes were shown to contain Fos family members. Down-regulation of PKC inhibited both the acute PDB-induced and the RA-induced increase in AP-1 activity. The selective PKC enzyme inhibitor, bisindolylmaleimide, reduced PDB-stimulated AP-1 activity, but enhanced RA-induced AP-1 activity. These results together with our previous studies suggest the intriguing possibility that PKC protein, but not enzyme activity, may be required for RA-induced AP-1 activity.

Topics: Animals; Cell Differentiation; Cells, Cultured; Enzyme Activation; Isoenzymes; Keratolytic Agents; Melanoma; Mice; Protein Kinase C; Protein Kinase C-alpha; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Transcription Factor AP-1; Transcription, Genetic; Tretinoin

Retinoic acid (RA) induces growth inhibition, differentiation or cell death in many human neuroblastoma cell lines. Recently, the transactivation activity of nuclear retinoids receptors has been shown to be modulated through physical association with other proteins that act as co-activators or as co-repressors. We investigated the expression of the co-repressor (SMRT) and co-activator (Trip 1) for retinoid and thyroid-hormone receptors in several neuroectodermal tumour cell lines, and its modulation by all-trans-retinoic acid, as well as by synthetic agonists, for RAR alpha, RAR beta, RAR gamma and RXR. We demonstrate that (i) SMRT and Trip-1 mRNAs are expressed in many human neuroblastoma and melanoma cell lines in basal conditions, (ii) SMRT mRNA expression in human neuroblastoma cell line SK-N-BE(2) increases after 48 hours of incubation with 1 microM RA and RARs specific agonists, (iii) Trip-1 mRNA in the same cell line does not change during incubation with RA or selective synthetic agonists for RARs and RXR.

Topics: Chromatography, High Pressure Liquid; Dimerization; DNA Primers; DNA-Binding Proteins; Electrophoresis, Agar Gel; Eukaryotic Initiation Factor-3; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Neuroblastoma; Nuclear Receptor Co-Repressor 2; Polymerase Chain Reaction; Proteins; Receptors, Retinoic Acid; Receptors, Thyroid Hormone; Repressor Proteins; RNA, Messenger; Transcriptional Activation; Tretinoin; Tumor Cells, Cultured

The hyaluronan-rich matrix that surrounds many tumours and facilitates tumour cell growth and invasion is thought to be predominantly synthesized by normal stromal cells stimulated by tumour cell-derived factors. This study examines the possibility that the production of tumour cell-derived factors that stimulate fibroblast glycosaminoglycan (GAG) synthesis may be blocked by exposure to differentiation-inducing agents such as retinoic acid. We have demonstrated that Hs294T, C8161 and A375 human melanoma cell lines release factors into their medium that stimulate normal fibroblast GAG synthesis. Exposure of these melanoma cells to retinoic acid failed to mediate any significant reduction in growth over a 7-day period. Retinoic acid failed to block the tumour cell production of GAG-stimulating activities and even enhanced the activities produced by the C8161 cell line, particularly at low retinoic acid concentrations (48% stimulation at 10(-9) M retinoic acid; P < 0.02). Addition of retinoic acid directly to fibroblast cultures exposed to fibroblast-conditioned medium resulted in an inhibition of GAG synthesis with a 33% inhibition observed at 10(-5) M. Addition of retinoic acid to fibroblast cultures exposed to the tumour cell-conditioned medium failed to inhibit the stimulation of GAG synthesis. Other differentiation-inducing agents, such as hexamethylene-bis-acetamide and butyrate, also failed to block the production of tumour cell-derived GAG-stimulating activities. These results demonstrate that retinoic acid and other differentiation-inducing agents fail to inhibit melanoma cell production of fibroblast GAG synthesis-stimulating factors or their action upon fibroblasts.

Topics: Acetamides; Adult; Antineoplastic Agents; Butyrates; Butyric Acid; Cell Count; Cell Differentiation; Cells, Cultured; Culture Media, Conditioned; Female; Fibroblasts; Glycosaminoglycans; Humans; Male; Melanoma; Middle Aged; Skin Neoplasms; Tretinoin

Retinoids inhibit proliferation of melanocytes and melanoma cells and affect disorders of hypo- and hyperpigmentation. Such effects might involve retinoid-binding proteins, retinoid metabolites and nuclear retinoid receptors for transcriptional activation. We detected messenger RNA transcripts for the cellular retinol- and retinoic acid-binding proteins (CRBP, CRABP I and II) in cultured epidermal melanocytes. In the melanoma cell lines the major transcript was CRABP II. Nuclear retinoic acid (RA) receptor transcripts and the 9-cis-retinoic acid receptor transcript were detected in all cells. The endogenous concentrations of retinol (ROH) and its metabolite 3,4-didehydroretinol (ddROH) in melanocytes were five times those in melanoma cells. When cells were incubated with [3H]ROH the main metabolites in the melanocytes were [3H]ddROH (4%) and [3H]RA (0.4%). Formation of [3H]RA was only detected in one melanoma cell line. Both melanocytes and melanoma cells produced an unidentified metabolite when incubated with [3H]ROH and [3H]RA. Dissimilarities in the metabolism and endogenous concentration of retinoids between benign and malignant melanocytes might play a key role in differentiation and growth regulation.

Topics: Cells, Cultured; Child; Gene Expression; Humans; Infant; Infant, Newborn; Male; Melanocytes; Melanoma; Polymerase Chain Reaction; Receptors, Cell Surface; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; RNA, Messenger; Skin; Tretinoin; Tritium; Tumor Cells, Cultured; Vitamin A

Retinoic acid (RA) induces growth arrest and differentiation of many different tumor cells. RA activates RA receptors, which function as ligand-dependent transcriptional modulators. S91 murine melanoma cells stop proliferating and then reversibly differentiate into a melanocytic cell type after the administration of RA. The genetic changes that take place during this process serve as an excellent model for the etiology of melanoma. The use of subtractive hybridization techniques yielded several differentially expressed cDNAs that are associated with RA-induced growth arrest. One clone, cyclin D1, is repressed and is probably a differentiation marker. Two other cDNAs represent novel, RA-inducible genes. Expression of another cDNA, clone 10d, is strongly down-regulated. It is the homologue of the human gene BM28 (CDCL1) that is indispensable for entry into S phase and cell division. S91 cells that are permanently transfected with a plasmid that constitutively expresses clone 10d become significantly more resistant to RA, suggesting that repression of this gene is a critical event in RA-induced growth arrest. The use of reverse transcription-PCR for the detection of expression in human melanoma in vitro was performed to study the potential role of clone 10d/BM28 in this disease. It is expressed in 80% of melanoma cell lines but is virtually undetectable in primary melanocytes. The expression of BM28 is not regulated by RA in human, RA-resistant melanoma cells. These results suggest that clone 10d/BM28 functions as an important tumor cell growth promoter. The regulation of clone 10d may be directly mediated by RA receptors, and escape from negative regulation may, thus, contribute to the etiology of melanoma.

Topics: Base Sequence; Cell Cycle Proteins; Cell Division; Cyclin D1; DNA, Complementary; DNA, Neoplasm; Down-Regulation; Drug Resistance, Neoplasm; Humans; Melanoma; Minichromosome Maintenance Complex Component 2; Molecular Sequence Data; Nuclear Proteins; Receptors, Retinoic Acid; Sequence Analysis, DNA; Tretinoin; Tumor Cells, Cultured

Solid tumors are relatively resistant to growth inhibition by IFNs. To enhance sensitivity, we assessed combinations of IFNs with all-trans-retinoic acid (RA). Antiproliferative studies in vitro suggested that the growth of three human breast carcinomas (MCF-7, MDA-MB-231, and MDA-MB-468), an ovarian carcinoma (NIH-OVCAR-3), and a malignant melanoma (SK-MEL-1) was inhibited to a greater degree by combination treatment with human IFN-beta and RA compared to single agents. Some of these cell lines were resistant to 10-100 IU/ml human IFN-alpha2b or IFN-beta or to 0.1-1.0 microM RA. Growth was inhibited significantly by combinations of IFNs and RA in all cell lines tested, and in some cases, cytotoxicity was observed. Sequential treatment of MCF-7 cells with RA followed by IFN-beta was more effective at inhibiting growth than treatment with IFN-beta followed by RA, suggesting that RA modulated the anticellular response of IFN-beta rather than the converse. In nude mice, the growth of MCF-7 and NIH-OVCAR-3 tumors was suppressed completely when combination treatment was started 2 days after tumor inoculation. Established, 6-week-old NIH-OVCAR-3 tumors underwent regression when treated with the combination of IFN-beta and RA but not with single-agent therapy. Together with our recent studies that demonstrated enhancement of IFN-stimulated gene expression by RA pretreatment in IFN-resistant cells, these data suggest that combination treatment with RA and IFNs may increase IFN-stimulated gene expression in IFN-resistant tumors, leading to augmented antitumor effects.

Topics: Animals; Breast Neoplasms; Cell Division; Combined Modality Therapy; Drug Synergism; Estradiol; Female; Humans; Interferon beta-1a; Interferon beta-1b; Interferon-beta; Melanoma; Mice; Mice, Nude; Ovarian Neoplasms; Receptors, Estrogen; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured

Induction of interleukin-8 (IL-8) by IL-1 or tumor necrosis factor (TNF), and repression by interferons or glucocorticoids have been shown to involve sequences between nucleotides -94 and -71 of the 5'-flanking region, and the transcription factors NF-IL-6 and NF-kappaB. The A3 cell line was derived from the human melanoma cell line G-361 by stable transfection with part of the IL-8 promoter (nucleotides -101 to +40 from transcription start) fused to the luciferase coding region. These regulatory sequences were sufficient for transcriptional activation by all-trans-retinoic acid (ATRA), 9-cis-retinoic acid, IL-1beta, or TNF-alpha. Simultaneous treatment of A3 cells with ATRA and TNF-alpha resulted in a dose- and time-dependent synergistic increase in luciferase expression and IL-8 mRNA levels. Transient transfections of the parental cell line demonstrated that the NF-kappaB binding site is essential for this synergistic transactivation. Electrophoretic mobility shift assays with nuclear extracts of A3 cells showed that stimulation with ATRA and TNF-alpha for more than 16 h resulted in enhanced NF-kappaB binding compared to that induced by TNF-alpha alone. The simultaneous treatment with ATRA and TNF-alpha also resulted in changes in the composition of NF-kappaB complexes bound to the IL-8 NF-kappaB site, preventing the formation of two TNF-alpha-inducible binding activities. We suggest that these complexes consist of repressive factors which, when removed, allow enhanced binding of NF-kappaB to its cognate site.

Topics: Drug Synergism; Humans; Inflammation Mediators; Interleukin-8; Melanoma; NF-kappa B; Promoter Regions, Genetic; Protein Binding; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Up-Regulation

B16 melanoma cells differentiate upon treatment with retinoic acid (RA). This differentiation process is accompanied by an increase of protein kinase C alpha (PKC alpha) mRNA and protein levels. Overexpression of PKC alpha in these cells results in a more differentiated phenotype, suggesting the importance of this protein in the control of differentiation by RA. The purpose of the study reported here was to determine the subcellular distribution of the RA-induced PKC alpha, whether the RA-induced increase in PKC alpha protein levels was accompanied by an increase in in situ enzyme activity, and whether RA altered AP-1 transcriptional activity. We found that RA treatment increased PKC alpha protein levels in all subcellular compartments examined, but it also induced a selective enrichment in nuclear-associated PKC alpha levels. Treating cells with an active phorbol ester induced translocation of PKC alpha to membrane fractions, but had no effect on nuclear PKC alpha levels. RA also increased PKC enzymatic activity in intact cells as determined by phosphorylation of the PKC-specific endogenous substrate MARCKS. However, while RA induced a five- to eightfold increase in total cellular PKC alpha protein levels, it only increased MARCKS phosphorylation by twofold. In light of the increase in in situ PKC enzyme activity and the enrichment of nuclear PKC alpha, we determined whether AP-1 activity might be increased in RA-treated cells. Use of luciferase reporter gene constructs with or without AP-1 elements transfected into B16 cells indicated that RA induced a four- to fivefold increase in AP-1 transcriptional activity. These results suggest a hypothesis whereby RA-induced nuclear PKC alpha might lead to increased AP-1 activity and show that RA-induced growth inhibition and differentiation are not always accompanied by an inhibition of AP-1 activity as has been proposed by other investigators.

Topics: Animals; Cell Differentiation; Cell Fractionation; Cell Membrane; Cell Nucleus; Intracellular Signaling Peptides and Proteins; Isoenzymes; Luciferases; Melanoma; Membrane Proteins; Mice; Myristoylated Alanine-Rich C Kinase Substrate; Phosphorylation; Protein Kinase C; Protein Kinase C-alpha; Proteins; Recombinant Fusion Proteins; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Invasion of basement membranes is a key step in systemic spread of tumour cells. To analyze genetic mechanisms involved in this process, we have selected strongly and weakly invasive sublines with stable phenotypes from a primary human melanoma cell line by repeated passage through a reconstituted basement membrane in vitro. The sublines differed approximately 5-fold in their invasive potential. Invasiveness correlated with better attachment and overexpression of the integrin alpha v/beta 3 (vitronectin/laminin-receptor). Treatment with retinoic acid inhibited proliferation in both sublines and invasion in the weakly invasive cells but stimulated invasion in the strongly invasive subline. Northern-blot analyses revealed equal levels of mRNA expression regarding collagenase type-IV and retinoic-acid receptors but enhanced expression of TIMP-2 mRNA in weakly invasive cells. The 2 sublines differed significantly in their respective DNA ploidy when compared to the wild-type Mel Im cell line, suggesting that they represent heterogeneous clones present in the primary tumour. We have started to exploit this in vitro system for tumour heterogeneity to clone genes involved in invasion. By a subtractive cDNA cloning strategy, 12 partial cDNA clones were obtained that are specifically overexpressed in the strongly or weakly invasive subline. These results illustrate that stable genetic alterations lead to heterogeneous subpopulations within primary melanomas which differ in their ability to invade basement membranes and interact with components of the extracellular matrix.

Topics: Actins; Aneuploidy; Clone Cells; Cloning, Molecular; Collagen; Collagenases; Disease Progression; DNA, Complementary; DNA, Neoplasm; Drug Combinations; Gene Expression Regulation, Neoplastic; Humans; Integrins; Laminin; Melanoma; Neoplasm Invasiveness; Neoplasm Proteins; Protein Biosynthesis; Proteins; Proteoglycans; Receptors, Cytoadhesin; Receptors, Vitronectin; Selection, Genetic; Skin Neoplasms; Tissue Inhibitor of Metalloproteinase-2; Tretinoin; Tumor Cells, Cultured

Previous data showing the correlation of multidrug resistance (MDR) and differentiation in tumor cell populations (Melloni et al. 1988; Stavrovskaya et al. 1990) suggest that: 1) isolation of MDR cells by cytostatic drugs leads to the selection of more differentiated cell variants and 2) in more differentiated cell variants the activity of MDR-related P-glycoprotein (Pgp) is more prominent than in less differentiated cells. Here we used human melanoma cell line mS and two variants selected from mS population: a) MDR variant of mS selected by colchicine (mS-0.5) and b) mS-trRAR/2--variant obtained by introduction of expressing retinoic acid receptor RAR-alpha cDNA into mS cell. The differentiation status, expression of MDR1 gene and Pgp functioning were compared in wild-type cells and mS variants. Electron microscopic examination of melanosomes showed that the mS-0.5 subline comprised more differentiating cells in the population than parental mS cultures and that these cells were at later stages of melanogenesis. The increase in the degree of differentiation in mS-0.5 population coincided with MDR1 gene overexpression, occurrence of Pgp molecules on the cell membrane and acceleration of Pgp-mediated Rhodamine 123 (Rh123) efflux. mS-trRAR/2, proved to be more differentiated than mS cells. The MDR1 mRNA level and Rh123 efflux were not elevated in mS-trRAR/2 cells, however, retinoic acid (RA) treatment increased both the degree of differentiation and Rh123 efflux in mS-trRAR/2 to a greater extent than in mS cultures. Thus, the data obtained in this study are in favor of the suppositions mentioned above. The mechanisms of coordinated alterations of differentiation and Pgp activity in MDR cells are discussed.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Division; Drug Resistance, Multiple; Humans; Melanins; Melanoma; Receptors, Retinoic Acid; Transfection; Tretinoin; Tumor Cells, Cultured

Retinoids play an important role as differentiating agents in a variety of normal and neoplastic cells and have been reported to induce ICAM-1 levels in melanomas, a phenomenon that we confirm in this paper. The effects of retinoids on gene expression usually involve the binding of specific retinoic acid receptor trans-acting factors (RARs) with their ligands, which then interact with specific target sites, the retinoic acid responsive elements (RAREs) present in the promoters of responsive genes. In the case of ICAM-1, we have cloned and analyzed the proximal regulatory region of the human gene. We show that the ICAM-1 promoter is RA-inducible, that it contains a putative consensus RARE (GGGTCATCGCCCTGCC), which binds in vitro RAR alpha complemented with RXRs, and that mutation of the RARE abrogates promoter responsiveness to RA. These studies allow ICAM-1 to be added to the list of genes transcriptionally activated by RA acting through an RARE element.

Topics: Base Sequence; Binding Sites; Cell Line; Chloramphenicol O-Acetyltransferase; Consensus Sequence; DNA-Binding Proteins; Gene Expression; Humans; Intercellular Adhesion Molecule-1; Melanoma; Molecular Sequence Data; Mutagenesis; Neoplasm Metastasis; Promoter Regions, Genetic; Receptors, Retinoic Acid; Regulatory Sequences, Nucleic Acid; Restriction Mapping; Retinoic Acid Receptor alpha; Retinoid X Receptors; Transcription Factors; Transfection; Tretinoin; Tumor Cells, Cultured

The integrin family of adhesion receptors is likely to be important for tumor cell invasion and dissemination. We have studied the effects of the differentiating agents retinoic acid on integrin expression by the human melanoma cell line MeWo. Our results show that this agent inhibits cellular proliferation, increases melanin content and induces morphological changes in MeWo cells. Functionally, these alterations are associated with an enhanced adhesion to matrix protein vitronectin and higher levels of expression of vitronectin receptor on the cell surface. This is accompanied by increased levels of alpha v integrin mRNA. Although the mechanism by which retinoic acid regulates the expression of vitronectin receptor in MeWo cells needs further examination, this system may represent a good model for understanding the role of this receptor in melanoma progression, as well the molecular basis for retinoic acid therapy in these tumors.

Topics: Blotting, Northern; Cell Adhesion; Cell Division; Humans; Integrins; Melanins; Melanoma; Receptors, Cytoadhesin; Receptors, Vitronectin; Tretinoin; Tumor Cells, Cultured

MTS1 is a metastasis-associated gene highly expressed in high-metastasis tumors. Here we show that the expression of the suppressor gene p53 protein correlates with mts1 expression. In murine melanoma B16-F1 cells, alpha-melanocyte-stimulating hormone up-regulated mts1 and increased p53 positivity in immunohistochemical tests. In B16-ML8 cells, retinoic acid reduced mts1 expression together with a reduction of p53 positivity. The variation of p53 in association with mts1 gene expression suggests that the mts1 product might interact with and stabilize p53. Taxol-induced aneuploidy increased the proportion of G0G1 phase cells, increased p53 positivity, and down-regulated mts1. This suggests that mts1 transcription may have been negatively regulated, possibly on account of the stabilization of microtubules by taxol. We postulate that the control of G1-S transition by p53 could be due to p53 sequestration by mts1, leading to microtubule depolymerization and signaling entry, into the S phase. Thus, a coordinated function of mts1 and p53 may be involved not only in uncontrolled growth but also in cytoskeletal depolymerization that could lead to cancer invasion.

Topics: Calcium-Binding Proteins; Cell Cycle; Gene Expression; Melanocyte-Stimulating Hormones; Melanoma; Neoplasm Metastasis; Paclitaxel; S100 Proteins; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

In an attempt to define the role of plasminogen activator in invasiveness and differentiation of human melanoma cells, the modulation of these parameters was studied in two melanoma clones characterized by marked differences in their basal features, using 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and retinoic acid, two differentiation inducers, and doxorubicin, a cytotoxic agent. TPA induced only slight reductions, whereas retinoic acid and doxorubicin caused an increase in invasiveness, enzymatic activity and differentiation in the clone showing low invasivity, low urokinase-type plasminogen activator levels and high differentiation. In contrast, in the clone showing high invasivity, high urokinase-type plasminogen activator levels and low differentiation it was found that: TPA was ineffective; retinoic acid induced a reduction of plasminogen activator but no modifications of invasiveness and differentiation; doxorubicin caused a decrease in invasiveness and plasminogen activator activity but no modification of morphological features. The different behaviour of the two clones thus could be related to the basal features of the clones. The results reported here indicate that in the presence of these drugs the associations between invasiveness and urokinase-type plasminogen activator activity and between invasiveness and differentiation are lost. Drug treatment therefore significantly affected the features of the clone characterized by low biological aggressiveness (high differentiation, low invasiveness), whereas the highly aggressive clone did not show a consistent response to drug treatment.

Topics: Cell Survival; Cell Transformation, Neoplastic; Chemotaxis; Doxorubicin; Humans; Melanoma; Neoplasm Invasiveness; Plasminogen Activators; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

Neuroblastoma cell lines have been reported to contain two proopiomelanocortin (POMC) mRNA transcripts. We have now shown by immunocytochemistry and radioimmunoassay (RIA) that a number of neuroectodermally derived cell lines contain immunoreactive beta-endorphin although cell concentrations were not characteristic of any tumour type. To explore further the functional significance of beta-endorphin expression, we analysed neuroblastoma cell lines having intermediate (I), substrate adherent (S) and neuronal (N) phenotypes. No differences in cell beta-endorphin content were detected. However, the expression of POMC mRNA and of immunoreactive beta-endorphin was reduced within a few hours of treatment of these cell lines with retinoic acid. Culture of the cell lines in the presence of beta-endorphin resulted in small but significant increases in growth. Although the POMC gene is in the same chromosomal segment as N-myc, which is normally amplified in neuroblastoma, no corresponding amplification of POMC could be demonstrated. The data suggest that POMC gene products may contribute to the autocrine/paracrine growth of neuroectodermal tumours.

Topics: beta-Endorphin; Cell Line; Clone Cells; Ectoderm; Gene Expression; Glioblastoma; Humans; Immunohistochemistry; Melanoma; Neuroblastoma; Neuroectodermal Tumors, Primitive, Peripheral; Pro-Opiomelanocortin; Radioimmunoassay; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

AHNAK is a newly identified human gene notable for the exceptional size (c.a. 700 kD) and structure of its product, and for the repression of its expression in human neuroblastoma cells. Here we report the identification and partial characterization of the protein encoded by AHNAK. The protein is located principally (but not exclusively) in the nucleus and is phosphorylated on both serine and threonine. The abundance of the protein increases appreciably when cells withdraw from the division cycle, in response to either withdrawal of serum (fibroblasts) or differentiation (neuroblastoma cells). By contrast, the amount of phosphorylation appears to diminish in those settings. The considerable abundance and conjectured fibrous structure of AHNAK protein suggest a role in cytoarchitecture, but no function can yet be discerned.

Topics: Amino Acid Sequence; Blotting, Western; Carcinoma, Small Cell; Cell Differentiation; Cell Fractionation; Cell Nucleus; Fluorescent Antibody Technique; Humans; Immune Sera; Lung Neoplasms; Melanoma; Membrane Proteins; Molecular Sequence Data; Molecular Weight; Neoplasm Proteins; Neuroblastoma; Peptides; Phosphoproteins; Tretinoin; Tumor Cells, Cultured

Neuroectodermal tumours express hormones which are post-translationally processed and inactivated by the action of specific proteases and peptidases. The data reported here show the presence of a novel thermolysin-like metallo-endopeptidase activity in several human cell lines. The soluble fractions of neuroblastoma, melanoma and a glioblastoma tumour cell lines are able, with different degrees, to cleave the Ser12-Phe13 bond of a DVDERDVRGFAS decreases FLNH2 substrate. The inhibition pattern suggests a metallo-endopeptidase thermolysin-like character, with the involvement of thiol group(s), clearly distinct from neutral endopeptidase (NEP; EC 3.4.24.11). This metallo-endopeptidase activity is down regulated during retinoic acid(RA)-induced neuronal differentiation in the RA-sensitive SK-N-BE(2) cells but not in the RA-resistant BE(2)-M17 cells, suggesting that the down regulation is related to neuronal differentiation and not a direct effect of RA on the enzymatic activity.

Topics: Amino Acid Sequence; Animals; Cell Differentiation; Glioma; Humans; Kinetics; Melanoma; Metalloendopeptidases; Molecular Sequence Data; Neoplasms, Germ Cell and Embryonal; Neprilysin; Neuroblastoma; Oligopeptides; Recombinant Proteins; Skin; Substrate Specificity; Thermolysin; Tretinoin; Tumor Cells, Cultured; Xenopus laevis

Human epidermal melanocytes were treated with all-trans-retinoic acid (RA) and examined for adhesion to bovine serum albumin-, fibronectin- and laminin-coated culture dishes. Control and treated cells were also examined for motility into micropore filters coated with the same proteins. Treatment of the cells with 3 x 10(-6) M RA for 3-4 days resulted in inhibition of attachment to all three substrates. Decreased attachment was observed within 1.5 h. Inhibition of attachment was not due to toxicity because differences between control and treated cells disappeared by 18 h, when most of the cells (approximately 75%) were attached and spread on all three substrates. The same treatment that inhibited adhesion also reduced migration into the interstices of micropore filters coated with the same three proteins. In additional experiments, human and mouse melanoma cell lines were examined in place of normal melanocytes. RA treatment also blocked adhesion and motility of these cells. The malignant melanoma cells were less sensitive to RA than normal melanocytes in the adhesion assay but were equally sensitive in the motility assay. The ability of RA to inhibit melanocyte adhesion and motility as well as melanocyte growth could explain, in part, the capacity of retinoids to modulate melanocyte function in hyperpigmented skin lesions.

Topics: Animals; Cattle; Cell Adhesion; Cell Line, Transformed; Cell Movement; Fibronectins; Humans; Infant, Newborn; Laminin; Melanocytes; Melanoma; Serum Albumin; Skin Neoplasms; Tretinoin

The active component of the honeybee hive product propolis, caffeic acid phenethyl ester (CAPE), has been shown to display increased toxicity toward various oncogene-transformed cell lines in comparison with their untransformed counterparts (Su et al., 4: 231-242, 1991). This observation provides support for the concept that it is the transformed phenotype which is specifically sensitive to CAPE. In the present study, we have determined the effect of CAPE on the growth and antigenic phenotype of a human melanoma cell line, HO-1, and a human glioblastoma multiforme cell line, GBM-18. For comparison, we have also tested the effects of mezerein (MEZ), mycophenolic acid (MPA) and retinoic acid (RA), which can differentially modulate growth, differentiation and the antigenic phenotype in these human tumor cell lines. Growth of both cell lines was suppressed by CAPE in a dose-dependent fashion, with HO-1 cells being more sensitive than GBM-18 cells. The antiproliferative effect of CAPE was enhanced in both cell types if CAPE and MEZ were used in combination. Growth suppression was associated with morphological changes in H0-1 cells, suggesting induction of a more differentiated phenotype. CAPE also differentially modulated the expression of several antigens on the surface of the two tumor cell lines. These results suggest a potential role for CAPE as an antitumor agent, an antigenic modulating agent and possibly a differentiation inducing agent.

Topics: Antigens, Neoplasm; Antineoplastic Agents, Phytogenic; Caffeic Acids; Cell Differentiation; Cell Division; Cytotoxins; Diterpenes; Glioblastoma; Humans; Melanoma; Mycophenolic Acid; Phenotype; Phenylethyl Alcohol; Terpenes; Tretinoin; Tumor Cells, Cultured

Early recognition of melanoma is directly related to improvement in survival. Patients, therefore, must not only be educated in recognition of abnormal skin lesions, but also in proper skin examination, ultraviolet radiation protection, effect of drugs on the development of malignancies, and dietary means of promoting wellness and preventing disease. Basic patient instruction should begin at the time of diagnosis and should include all health care team members to assure patient comprehension and compliance with recommendations.

Topics: Dietary Fats; Dietary Fiber; Humans; Melanoma; Patient Education as Topic; Skin Aging; Skin Neoplasms; Sunscreening Agents; Survival Rate; Tretinoin

beta-All-trans-retinoic acid (RA) has been shown to inhibit the growth, enhance the differentiation, and suppress the transformed and metastatic properties of certain human and murine melanoma cells. This study examined the effect of RA on the level of a cell surface receptor (M(r) 78,000) (gp78) for an autocrine motility factor, which has been implicated in invasion and metastasis. Treatment of murine melanoma cell lines S91-C2, B16-F1, and K1735-P with RA (10 microM) for 5 days decreased the level of gp78 by 37, 72, and 92%, respectively, as revealed by immunoblotting with monoclonal antibodies raised against gp78. In contrast, RA had only a limited effect on gp78 levels in melanoma cell clones or variant cell lines that are resistant to the growth-inhibitory effects of RA (S91-C154, B16-F10, and K1735-Cl19). Further studies with K1735-P, the most sensitive cell line with respect to modulation of gp78, showed that the decrease in gp78 level required at least 1 microM RA and 4 to 5 days of treatment. The binding of anti-gp78 antibodies to the surface of intact RA-treated cells and to intracellular gp78 in permeabilized cells was also lower than in untreated cells. Furthermore, RA treatment decreased the induction of cell motility, on colloidal gold-coated glass coverslips, by anti-gp78 antibodies, which mimic the effect of autocrine motility factor. The RA-induced decrease in antibody-enhanced cell motility was similar to the time- and RA concentration-dependent decrease in the amount of gp78, suggesting that the two events are related. These results raise the possibility that the previously reported suppression by RA of tumor cell invasion and metastasis may be related, at least in part, to suppression of cell motility resulting from the decreased level of the autocrine motility factor receptor.

Topics: Animals; Antibodies, Monoclonal; Cell Movement; Down-Regulation; Glucose-6-Phosphate Isomerase; In Vitro Techniques; Melanoma; Mice; Receptors, Autocrine Motility Factor; Receptors, Cell Surface; Receptors, Cytokine; Tretinoin; Tumor Cells, Cultured; Ubiquitin-Protein Ligases

The potential role of intercellular adhesion molecule 1 (ICAM-1) in the biology of human melanoma cells has stimulated interest in the characterization of its modulation. The present study has shown that the differentiating agent retinoic acid (RA) up-regulates ICAM-1 expression by melanoma cells in a dose- and time-dependent fashion. The enhancement of ICAM-1 cell surface expression is paralleled by an increase in ICAM-1 mRNA. Therefore, ICAM-1 represents an additional gene which may be transcriptionally regulated by RA. The five melanoma cell lines tested displayed a differential susceptibility to the modulation of ICAM-1 expression by RA, since the cell line MeWo did not change in its ICAM-1 expression following incubation with RA. Nevertheless, RA-insensitive as well as RA-sensitive melanoma cell lines displayed a higher increase in ICAM-1 expression following incubation with RA and cytokines than following incubation with each of them. Analysis of the distribution in the melanoma cell lines of retinoic acid receptors (RARs) showed a relationship between susceptibility to a RA-mediated increase of ICAM-1 expression and RAR beta expression, suggesting that the latter receptor may play a role in the phenomenon. RAR alpha and RAR gamma were present in RA-sensitive and -insensitive melanoma cell lines, suggesting that they play a role in the enhancement by RA of cytokine-mediated up-regulation of ICAM-1 expression. The melanoma cell lines we have described may represent a useful system for investigating the role of RAR in the regulation of gene expression and the mechanism(s) which underlie this effect.

Topics: Carrier Proteins; Cell Adhesion Molecules; Gene Expression; Humans; In Vitro Techniques; Intercellular Adhesion Molecule-1; Interferon-gamma; Interleukin-1; Melanoma; Receptors, Retinoic Acid; RNA, Messenger; Time Factors; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Human epidermal melanocytes were examined for proliferation under various conditions in the presence or absence of all-trans retinoic acid (RA). Under conditions which supported proliferation, RA at concentrations of 0.25-1.0 microgram/ml inhibited cell growth but was not cytotoxic. When melanocytes were cultured under conditions which by themselves did not support growth, RA did not overcome the growth limitation. Treatment of melanocytes with RA altered their morphological appearance. Alterations included retraction of dendritic processes, increased flattening, and a slight darkening of the cytoplasm in some of the cells. However, when examined biochemically, there was no significant change in the amount of malanin per cell or in tyrosinase activity. RA also inhibited proliferation of six different malignant melanoma lines. Inhibition was observed over the same RA concentrations and over the same time course in the melanoma cells as was seen in melanocytes. Inhibition of melanocyte and melanoma cell proliferation was slowly reversed following removal of RA from the culture medium. These results indicate that RA can inhibit proliferation of melanocytic cells.

Topics: Animals; Cell Division; Cells, Cultured; Dendrites; Dose-Response Relationship, Drug; Humans; Male; Melanins; Melanocytes; Melanoma; Mice; Tretinoin; Tumor Cells, Cultured; Tyrosine

Two lines of murine melanoma cells (B16 and Cloudman S91) were cultured on type I collagen gel and the effects of all-trans-retinoic acid on the growth and infiltration into the gel were assayed. In both lines, proliferation and the degree of infiltration were suppressed by the addition of all-trans-retinoic acid. The infiltration-inhibiting effect was expressed very rapidly and was dose-dependent at concentrations ranging from 10(-7) to 10(-5) M of all-trans-retinoic acid. These results suggest the anti-invasive effects of all-trans-retinoic acid on melanoma cells.

Topics: Animals; Cell Division; Collagen; Dose-Response Relationship, Drug; Gels; Melanoma; Mice; Neoplasm Invasiveness; Skin Neoplasms; Tretinoin; Tumor Cells, Cultured

The ability of retinoic acid to inhibit the growth of three human melanoma cell lines (MEW18, MEW22, MEW81) was studied in culture. The exposure of the cell lines to different concentrations of RA resulted in an inhibition of the cell growth, dependent on the quantity of RA in the medium. Effects on cell growth of MEW81 cells by RA, introduced into the medium in DMSO solution or incorporated in MLV, were also compared. The inhibitory potencies of the drug in two forms were similar, however, the inhibitory effects of RA in MLV were more stable than the effects of RA dissolved in DMSO.

Topics: Cell Division; Dimethyl Sulfoxide; Drug Carriers; Humans; Liposomes; Melanoma; Tretinoin; Tumor Cells, Cultured

The dispersed neuroendocrine system includes cells with different embryological derivations, sharing a common neuroendocrine (NE) program, as indicated by the expression of NE markers, some of which are shared antigenic determinants. We report here that the small cell lung carcinoma cells NCI-H69 express the two human melanoma-associated antigens (HMAA) NGA/LS62 an LS109. Incubation of NCI-H69 cells with maturational inducers, such as retinoic acid and bromodeoxyuridine (BrdU), upregulated the expression of both HMAA. Exposure to BrdU for 4 weeks induced the appearance of a different phenotype in subpopulations of NCI-H69 cells, which became epithelioid, substrate-adherent, grew in monolayer and continued to express NE-associated antigens in variable amount. The shift in phenotype was not reversible after BrdU withdrawal and was maintained for at least 6 months in continuous culture. The substrate adhesion of NCI-H69 cells was paralleled by a change in NGA glycosylation pattern, thus suggesting a possible functional role for NGA in cell substrate adhesion/recognition.

Topics: Antigens, Neoplasm; Bromodeoxyuridine; Carcinoma, Small Cell; Cell Adhesion; Flow Cytometry; Humans; Lung Neoplasms; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Precipitin Tests; Tretinoin; Tumor Cells, Cultured

We have described two human melanoma-associated antigens (HMAA), recognized by the murine monoclonal antibodies LS62 and LS109. LS62 recognizes the neuroglandular antigen (NGA), which is overexpressed in neoplastic melanocytes as well as in several tissues of neuroectodermal origin. These antibodies were used to screen six neuroblastoma cell lines and one neuroepithelioma cell line. A melanoma cell line, G361, known to express the two antigens, was used as the positive control. Variable expression of the two antigens was detected in neuroblastoma cells. The surface expression of NGA and of the LS109 antigen was modulated in parallel with the morphological differentiation induced by retinoic acid, 5-bromodeoxyuridine, or cyclic AMP analog/activators. The modulation of the expression of the two HMAA was detected in G361 melanoma cells and in one of the neuroblastoma cell lines, SK-N-SH. These results suggest altered expression of both antigens during melanoma and neuroblastoma cell differentiation in culture.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Bromodeoxyuridine; Cell Differentiation; Cyclic AMP; Flow Cytometry; Humans; Immunohistochemistry; Melanoma; Mice; Neuroblastoma; Precipitin Tests; Tretinoin

Topics: Administration, Topical; Humans; Melanoma; Skin Neoplasms; Tretinoin

To study the induction of differentiation in human melanoma cells, we treated 12 melanoma cell lines with mycophenolic acid and tiazofurin, inhibitors of IMP dehydrogenase (IMPDH). In all cell lines studied, both agents inhibited cell growth and increased melanin content. However, the degree of growth inhibition did not necessarily correspond to the increase in melanin content. A detailed analysis of the HO and SK-MEL-131 cell lines indicated that mycophenolic acid and tiazofurin caused a time- and dose-dependent increase in the expression of a series of other maturation markers, including formation of dendrite-like structures, tyrosinase activity, and reactivity with the CF21 monoclonal antibody. The growth inhibition and melanogenesis induced by the IMPDH inhibitors was abrogated by the addition of exogenous guanosine. No such effect was observed after treatment of the cells with phorbol 12-myristate 13-acetate or retinoic acid, two other inducers of differentiation in these cells. The mycophenolic acid- and tiazofurin-treated cells also showed an increased level of IMPDH mRNA and protein, perhaps because of compensation for the inhibitor-mediated decrease in IMPDH activity. In contrast, treatment with phorbol 12-myristate 13-acetate or retinoic acid resulted in decreased levels of IMPDH mRNA and protein. The lack of a consistent pattern of IMPDH expression in the cells treated with IMPDH inhibitors and phorbol 12-myristate 13-acetate or retinoic acid suggests that the altered expression of IMPDH is not a general requirement for the induction of cell differentiation in these cells. Our results also suggest that IMPDH inhibitors may provide a useful approach to circumvent the differentiation block in melanoma.

Topics: Biomarkers; Gene Expression Regulation, Neoplastic; Guanosine; Humans; IMP Dehydrogenase; Melanins; Melanoma; Monophenol Monooxygenase; Mycophenolic Acid; Neoplasm Proteins; Ribavirin; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

beta-All-trans retinoic acid (RA) treatment of murine S91-C2 melanoma cells decreases in vitro growth and modulates the glycosylation of specific cellular and cell-surface glycoproteins. The effect of RA treatment on [3H]fucose, [3H]galactose, and [3H]glucosamine incorporation was investigated by metabolic labeling followed by analysis of labeled cellular glycoproteins using polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) and fluorography. RA treatment dramatically increased the incorporation of the labeled monosaccharides into one glycoprotein of Mr 160,000 (gp160), which has been previously implicated in the growth-inhibitory effect of RA on these cells. Following RA treatment, cell-surface sialic acid residues on gp160 were also more intensely labeled by NaIO4 oxidation and subsequent NaB[3H]4 reduction than were those on gp160 of untreated cells. The activities of fucosyl- and galactosyltransferase increased about 1.5 to 1.9 times after RA treatment. These results suggest that the increased activities of the two glycosyltransferases is responsible for the increased incorporation of fucose and galactose into gp160.

Topics: Animals; Cattle; Culture Media; Fucose; Fucosyltransferases; Galactose; Galactosyltransferases; Glycopeptides; Glycoproteins; Glycosylation; Melanoma; Mice; Tretinoin; Tumor Cells, Cultured

Treatment of four A375 human melanoma sublines (A375, A375P, A375P-5, A375M), exhibiting distinct metastatic potentials in vivo, with beta-all-trans-retinoic acid in vitro caused a dose- and time-dependent inhibition of the ability of these cells to penetrate Matrigel-coated filters using a reconstituted basement membrane invasion assay. The possible mechanisms of action responsible for the antiinvasive effect were further investigated, and the data showed that compared with untreated cells the retinoic acid-treated cells: (a) secreted lower levels of collagenolytic enzymes, as demonstrated by a decreased ability of the cells to degrade [3H]proline-labeled type IV collagen substrate and by a reduction in the activity of a secreted Mr 64,000 collagenolytic enzyme detected in type IV collagen-containing polyacrylamide gels; (b) expressed lower levels of the human type IV collagenase mRNA (except in the A375P cells), as detected by Northern blot analysis; (c) exhibited decreased levels of tissue plasminogen activator activity, as demonstrated by a chromogenic assay; (d) were 10-40% less adhesive to a reconstituted basement membrane matrix, as determined by a 60-min Na2(51)CrO4-labeled cell attachment assay; (e) exhibited an increase in the high affinity metastasis-associated cell surface laminin receptor, as determined by flow cytometry after binding of fluorescently labeled laminin receptor antibody; and (f) expressed decreased amounts of gp78, a cell surface receptor for motility factor, demonstrated by immunoblotting and immunofluorescence. Collectively, these data suggest that retinoic acid inhibits tumor cell invasion through a basement membrane-like matrix by suppressing matrix degradation and by altering cell surface receptors.

Topics: Basement Membrane; Cell Adhesion; Cell Division; Dose-Response Relationship, Drug; Enzyme Induction; Humans; Melanoma; Microbial Collagenase; Neoplasm Invasiveness; Neoplasm Metastasis; Plasminogen Activators; Receptors, Cell Surface; Receptors, Immunologic; Receptors, Laminin; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

The activity of liposome-associated retinoic acid was analyzed on in vitro cultured tumor cell lines and compared to the antiproliferative effects of free retinoic acid. It was found that liposome-associated retinoic acid is about 300 times more active than free retinoic acid in inhibiting in vitro cell growth of leukemic and melanoma cell lines. An increased activity of retinoic acid (10-20 times) was also obtained after premixing of this compound with empty liposomes, demonstrating that the retinoic acid efficiently interacts with liposomes which may facilitate solubility and cell uptake of retinoids.

Topics: Animals; Cell Division; Drug Carriers; Freeze Fracturing; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Liposomes; Melanoma; Mice; Microscopy, Electron; Phosphatidylcholines; Tretinoin; Tumor Cells, Cultured

Treatment of a human cell line (HXG-2), established from a metastatic melanoma, with retinoic acid (RA) induced morphologic differentiation and eliminated its cloning capacity in soft agar. With the v-erb B oncogene as a probe, slot blot hybridization of genomic DNA from parental HXG-2 cells did not show epidermal growth factor (EGF) receptor gene amplification as compared with normal diploid fibroblasts. Analysis of RNA as well as EGF receptor determinations from HXG-2 and RA-treated HXG-2 cells showed essentially no differences, indicating that RA treatment does not modulate EGF receptor gene expression. Although enhanced EGF receptor expression is found in some advanced-stage melanomas, RA-induced changes in the transformation phenotype of cell line HXG-2 probably do not result from modulation of the EGF-mediated pathway.

Topics: DNA Probes; DNA, Neoplasm; ErbB Receptors; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Nucleic Acid Hybridization; Oncogenes; RNA, Neoplasm; Tretinoin; Tumor Cells, Cultured

The effect of retinoic acid on the induction of tyrosinase (EC 1:14.18.1) by imidazole was determined in cultured B16/C3 melanoma cells. Retinoic acid could block the induction of enzyme activity within 3 hours of addition to the culture medium at a physiological concentration (10nM). The blockade was similar to that of 3,3',5-L-triiodothyronine (T3) already reported. mRNA hybridizable to a tyrosinase DNA probe was induced by imidazole while retinoic acid and T3 blocked that increase. These observations suggest that retinoic acid can mimic the action of T3 in B16 melanoma cells in culture.

Topics: Animals; Catechol Oxidase; Cell Line; Enzyme Induction; Imidazoles; Melanoma; Mice; Monophenol Monooxygenase; RNA, Messenger; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

Concurrent with a phase-II trial of 4HPR in patients with various cancers, we studied the plasma pharmacokinetics of both 4HPR and its major metabolite 4MPR as well as the effect of 4HPR administration on plasma retinol concentrations using a simple, specific and sensitive HPLC procedure. Initial estimates of plasma pharmacokinetic parameters after oral administration of 4HPR (300 mg/day) [corrected] in 3 cancer patients were the following: 4HPR, t beta 1/2 = 13.7 hr, AUC = 3.49 micrograms.hr/ml, CL = 56.57 L/hr/m2; 4MPR, t beta 1/2 = 23.0 hr, AUC = 1.15 micrograms.hr/ml, CL = 239.29 L/hr/m2. We also found that oral administration of 4HPR resulted in a rapid, profound and significant reduction in plasma retinol concentrations. The mean plasma retinol concentrations for 9 patients decreased 60% from baseline to below 200 ng/ml within 1-2 weeks of 4HPR dosing initiation. In addition, there was a concurrent, significant reduction in plasma retinol-binding protein levels in these patients. The mechanism whereby 4HPR reduces plasma retinol levels in vivo has not been determined. The addition of 4HPR to pooled human plasma at 37 degrees C in vitro did not reduce endogenous retinol levels, suggesting no direct chemical interaction between these 2 retinoids.

Topics: Administration, Oral; Breast Neoplasms; Chromatography, High Pressure Liquid; Drug Evaluation; Fenretinide; Humans; Melanoma; Mycosis Fungoides; Retinol-Binding Proteins; Retinol-Binding Proteins, Plasma; Tretinoin; Vitamin A

Lectins purified by affinity chromatography on immobilized asialofetuin from extracts of mouse K-1735P melanoma cells appeared as two polypeptides [L-14.5 (Mr 14,500) and L-34 (Mr 34,000)] in one-dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. However, in two-dimensional electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate:polyacrylamide gel electrophoresis) the L-14.5 polypeptide was resolved into three acidic forms of pI 4.6, 4.9, and 5.8, whereas the L-34 was resolved into two polypeptides of pI 4.9 and 5.3. Antibodies directed against galactoside-binding lectins from rat and bovine lungs, mouse 3T3 fibroblasts, and mouse UV-2237 fibrosarcoma cells reacted with the K-1735P lectins in immunoblots, and normal mouse lung extracts were found to contain cross-reactive proteins that comigrated with the two melanoma lectins. Indirect immunofluorescence staining using the above antibodies demonstrated that both L-14.5 and L-34 were expressed on the surface of viable K-1735P cells. Treatment of these cells with 1 microM beta-all-trans-retinoic acid or 1 mM N6,O2'-dibutyryl cyclic AMP for 5 days induced morphological differentiation, inhibition of anchorage-dependent and anchorage-independent growths, and a selective decrease in the L-34 lectin level. Growth inhibition by starvation for serum factors, which did not induce differentiation, had no effect on the level of L-34. These results demonstrate that the melanoma lectins are immunologically related to normal cell lectins and that the two polypeptide species are expressed on the cell surface. Further, they demonstrate that the L-34 lectin level can be modulated by agents that suppress the transformed phenotype by enhancing differentiation.

Topics: Animals; Bucladesine; Cell Differentiation; Galectins; Hemagglutinins; Isoelectric Point; Melanoma; Mice; Mice, Inbred C3H; Molecular Weight; Tretinoin

Topics: Administration, Topical; Humans; Male; Melanoma; Middle Aged; Neoplasm Recurrence, Local; Skin Neoplasms; Tretinoin

Nude mice are deficient in thymus gland development and hence lacking functional, mature T-lymphocytes. Weanling nude mice were given various deficient and high retinyl palmitate (RP) or 13-cis retinoic acid (CRA) diets. The high RP (vitamin A) diets stimulated phagocytosis in the absence of mature T-helper cells. However, T-cell dependent mitogens did not cause significant mitogenesis in any group, while LPS, a B-cell mitogen, did. RP had no effect on mitogenesis. NK cell activity was increased only at a very high level of RP, as has been reported with conventional mice. Macrophage production of cytotoxic factors was unaffected by high levels of RP or CRA. Direct cytotoxicity in vitro of tumor cells was increased only at very high RP levels. Thus, mature T cells may be needed for RP to produce normal activation of macrophage, except at very high RP levels.

Topics: Animals; B-Lymphocytes; Cytotoxicity, Immunologic; Diterpenes; Female; Immunologic Deficiency Syndromes; Killer Cells, Natural; Macrophages; Melanoma; Mice; Mice, Nude; Mitogens; Phagocytosis; Retinyl Esters; T-Lymphocytes; Tretinoin; Vitamin A

Retinoic acid (RA) and butyric acid (BA) were investigated for their effect on in vitro migration of highly metastatic murine B16a melanoma cells. These potential antitumor agents are known to alter the cytoskeleton. Our initial studies determined the 72 h cytostatic/cytotoxic concentrations of RA (1 X 10(-6) M 1 greater than 1 X 10(-5) M) and BA (1.5 mM)/ greater than 2.0 mM). Cytostasis by RA and BA was confirmed by autoradiography and radioisotope incorporation. For migration assays, cells were plated on 3 and 5 microns diameter pore polycarbonate membranes. Complete media was added containing RA or BA at time of plating. For BA pretreatment studies, BA was added to cells for 72 h prior to plating cells in fresh BA on the membranes. Top and bottom surface of the membranes were examined after 72 h of incubation by scanning electron microscopy. Although RA and BA induced cells on top of the membrane to change morphology as shown by phase, transmission and scanning electron microscopy, only BA enhanced the deformability of cells to allow for passage through the 3 micron diameter pores. Butyric acid enhanced migration through 3 micron diameter pore membrane by 511%. For 5 micron diameter pore membranes, 55.2% of the plated number of untreated early passage cells migrated to the bottom surface as compared to 57.3% for BA-treated cells and 14.9% for RA-treated cells. However, if cellular proliferation over the 72 h period was factored in, BA increased migration by 456% over the controls and pretreatment of cells with BA for 72 h prior to plating increased migration by 893%. Without considering proliferation, RA inhibited migration by 75% over controls. The decrease in migration observed in RA-treated cells was due to an inhibitory effect on cellular migration and a decrease in proliferation.

Topics: Animals; Butyrates; Cell Line; Cell Movement; Cell Transformation, Neoplastic; Cytoskeleton; Melanoma; Mice; Microscopy, Electron; Microscopy, Electron, Scanning; Thymidine; Tretinoin; Tumor Cells, Cultured; Uridine

The melanocytotoxic effects of 4-hydroxyanisole (4-OHA) are thought to depend upon its conversion to toxic oxidation products by the enzyme tyrosinase. In this study, the cytotoxicity of 4-OHA was examined in different B16 melanoma cell lines that show varying levels of tyrosinase and after stimulation by melanocyte-stimulating hormone (MSH) and all-trans-retinoic acid (RA). 4-OHA decreased cell survival of three melanotic and one amelanotic cell line in culture, but the effect was unrelated to their tyrosinase activity or the subcellular localization of the enzyme. Although stimulation of tyrosinase activity with RA enhanced the cytotoxicity of 4-OHA, no similar enhancement occurred with alpha-MSH. It appears that there is no relationship between the cytotoxic effects of 4-OHA and intracellular tyrosinase and the enhancement of its cytotoxicity by RA may well be related to the antiproliferative effects of the retinoid.

Topics: Animals; Anisoles; Catechol Oxidase; Cell Survival; Ditiocarb; Melanocyte-Stimulating Hormones; Melanoma; Mice; Monophenol Monooxygenase; Tretinoin; Tumor Cells, Cultured

The effect of retinoic acid on murine B16 melanoma cell growth, tyrosinase activity and melanin synthesis was investigated. Retinoic acid inhibited the growth of B16F1, B16F10 and B16BL6 melanoma cells, but enhanced melanin synthesis only in the B16F1 cells. The B16F10 and B16BL6 cells exhibited retinoic acid-induced suppression of tyrosinase activity and melanin synthesis, which was most apparent in the B16F10 cell variant. For comparison, Cloudman S91 melanoma cells proved to be particularly sensitive to retinoic acid-induced growth inhibition and stimulation of the expression of their melanotic phenotype. These results suggest considerable heterogeneity in the B16 melanoma with respect to their response to retinoic acid.

Topics: Animals; Cell Division; Cell Line; Dose-Response Relationship, Drug; Melanins; Melanoma; Mice; Monophenol Monooxygenase; Tretinoin

The purpose of the experiments was to establish whether individual cells of a tumor cell population, or clonal lines derived from its express the differentiated phenotype, or respond heterogeneously following treatment with inducers of differentiation or with cytostatic drugs. The human cell lines used in this study were: HL-60 promyelocytic leukemia, K562 erythroleukemia, BHM-97 and A2058 melanoma, and A-1, A-2, A-4 and A-6 clones of A2058 line. Inducers of differentiation were phorbol myristate acetate (PMA), dimethylsulfoxide (DMSO) and retinoic acid (RA); cytostatics: adriamycin (ADM), 5-fluorouracil (5-FU), dacarbazine (DTIC), cis-platin (platidiam, PD) and arabinosyl cytosine (ara-C). Expression of the differentiated phenotype was shown by cell attachment (HL-60), hemoglobin production (K562), dendrit formation (A2058, BHM-97). Individual cells expressed the differentiated phenotype heterogeneously in all types of cell populations. Clone A-4 was the most, and clone A-6 the least sensitive to PMA. The drug sensitivity of the clones was different and drug-dependent. It is concluded that induction of differentiation as another approach to therapy of cancer, similar to anticancer drug therapy, also implies disadvantages due to population heterogeneity. Combinations of cytostatics with differentiation inducers might result in improved therapeutic effects.

Topics: Antineoplastic Agents; Cell Differentiation; Cisplatin; Cytarabine; Dacarbazine; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Doxorubicin; Fluorouracil; Humans; Leukemia, Erythroblastic, Acute; Leukemia, Promyelocytic, Acute; Melanoma; Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

A human cell line established from a metastatic melanoma had both multiple numerical and structural chromosome aberrations including one to two copies of a submetacentric marker chromosome with an insertion of an active nucleolar organizer region (NOR). Treatment of this cell line with retinoic acid (RA) induced morphologic differentiation and reduced the cellular saturation density concomitant with a significant decrease in Ag-NOR activity. RA-treated cells grown in the absence of this differentiating agent, however, displayed a return to normal Ag-NOR activity, indicating the effect of this chemical on ribosomal genes is reversible.

Topics: Animals; Cell Differentiation; Dimethyl Sulfoxide; Humans; Karyotyping; Melanoma; Mice; Mice, Nude; Nucleolus Organizer Region; Tretinoin; Tumor Cells, Cultured; Vitamin A

Previous studies have demonstrated the ability of retinoic acid (RA) to inhibit the growth of two spontaneous murine melanoma cell lines (B16-F1 and S91-C2) and to augment both sialyltransferase activity and the sialylation of an Mr 160,000 cell-surface glycoprotein. The present study examined the effects of RA on an ultraviolet irradiation-induced murine melanoma cell line K-1735P. Like the two spontaneous melanomas, the uv-induced melanoma exhibited susceptibility to the growth-inhibitory action of RA. Both the anchorage-dependent and the anchorage-independent growths of the K-1735P cells were suppressed by RA, with IC50 values of 5 X 10(-9) and 3 X 10(-12) M, respectively. Sialyltransferase activity in both S91-C2 and K-1735P cells treated with 10(-6) or 10(-5) M RA increased two- and three-fold, respectively, as compared with untreated cells. In contrast, cell-surface sialo- and galactoglycoproteins, revealed by labeling with periodate and tritiated borohydrate or with neuraminidase, galactose oxidase, and tritiated borohydrate, respectively, varied between the S91-C2 and the K-1735P cells, and each cell line's modulation by RA was also distinct. These findings suggest that although RA can increase the activity of sialyltransferase in different melanoma cells, this increased activity may, in turn, result in an increased sialylation of distinct cell-surface glycoproteins.

Topics: Animals; Cell Adhesion; Cell Line; Glycosylation; Melanoma; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Mice, Inbred DBA; Molecular Weight; N-Acetylneuraminic Acid; Sialic Acids; Sialyltransferases; Tretinoin

The ability of difluoromethylornithine (DFMO), a specific polyamine synthesis inhibitor, to interact with various biological modifiers to inhibit the colony-forming growth of human melanoma cells was determined by using the median effect principle to computer model the strength of two agent interactions. Either alpha- or gamma-IFN (interferon) in combination with DFMO resulted in a synergistic inhibition on human melanoma colony formation. For human melanoma cells which were not resistant to 13-cis RA (retinoic acid), an additive suppression on colony formation was obtained with the retinoid-DFMO combination. Dexamethasone (DEX) interacted with DFMO to yield a synergistic reduction in melanoma colony number on glucocorticoid sensitive cells and no growth enhancement with DFMO on glucocorticoid resistant melanoma lines. Human melanoma cells displayed differential long-term growth sensitivity to DFMO treatment. C8146C human melanoma cells were terminally growth-inhibited by a 96 h exposure to DFMO, in a manner which was concentration and time dependent. The proliferation of C82-7A melanoma cells was inhibited by 95% in presence of DFMO, but upon removal of DFMO the cells regained their ability to proliferate and form colonies. The simultaneous addition of DEX plus alpha-IFN plus 13-cis-RA with DFMO caused most of the human melanoma cells in these lines to become permanently growth arrested. Pre-treatment with DEX plus alpha-IFN plus 13-cis RA, but without DFMO, did not have any long term effect on the ability of melanoma cells to recover and proliferate in soft agar.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Division; Cell Line; Culture Techniques; Dexamethasone; Eflornithine; Humans; Interferon Type I; Melanoma; Recombinant Proteins; Tretinoin; Tumor Cells, Cultured

Previous studies have shown that treatment of S91-C2 murine melanoma cells with beta-all-trans-retinoic acid (RA) results in growth inhibition, enhanced activity of sialyltransferase, and increased glycosylation of a Mr 160,000 cell surface sialoglycoprotein (gp160). None of these effects could be detected in mutant clones (e.g., S91-C154) selected from the S91-C2 cells for resistance to RA-induced growth inhibition. These findings suggest that modulation by RA of gp160 might be related causally to growth inhibition. In this study we examined the possible role of gp160 in growth regulation using specific antibodies to this glycoprotein. Metabolic labeling of S91-C2 cells with either [3H]glucosamine or [35S]methionine revealed that the cells shed into the growth medium a gp160-like glycoprotein, in addition to several other macromolecules. The gp160-like glycoprotein was isolated from concentrated conditioned medium after preparative polyacrylamide slab gel electrophoresis in the presence of sodium dodecylsulfate by excision of the corresponding protein band. Rabbits were immunized with this material and immunoblotting revealed that their sera contained antibodies that bound specifically to gp160 in extracts of untreated or RA-treated S91-C2 cells. Indirect immunofluorescence staining followed by fluorescence-activated cell sorter analysis demonstrated that the anti-gp160 antibodies bound to the surface of both untreated and RA-treated S91-C2 cells and that the treated cells bound more of the antibodies than untreated ones. In contrast, these antibodies bound to the same extent to untreated and RA-treated resistant S91-C154 cells. The growth of S91-C2 cells in the presence of anti-gp160 antibodies in semisolid medium as well as in monolayer cultures was inhibited in a dose-dependent fashion. Fifty % growth inhibition was obtained at an immunoglobulin concentration of 10 micrograms/ml. The growth of cells exposed concurrently to RA and anti-gp160 antibodies was also inhibited strongly in semisolid medium, but the antibodies caused only a small increase in the inhibitory effect of RA in monolayer cultures. No inhibition by the antibodies of either anchorage-independent growth or anchorage-dependent growth of S91-C154 cells, grown in the absence or presence of RA, was observed. These results support the suggestion that cell surface gp160 might be involved in growth regulation in the S91-C2 cells.

Topics: Animals; Antibodies, Neoplasm; Dose-Response Relationship, Drug; Glucosamine; Glycoproteins; Immunosorbent Techniques; Melanoma; Methionine; Mice; Molecular Weight; Tretinoin

Cellular retinoic acid-binding proteins (CRABP) and cellular retinol-binding protein (CRBP) can be found in cells and nuclei. They function in the same way as receptors. CRABP and CRBP were studied in 9 cases of choroidal melanoma and in 3 of retinoblastoma. CRABP was found in 2 cases of melanoma and in 3 cases of retinoblastoma. CRBP was found in 1 melanoma.

Topics: Binding Sites; Eye Neoplasms; Humans; Melanoma; Osmolar Concentration; Retinoblastoma; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Tretinoin

Tumor-producing substances that promote lipolysis in vitro may also account for fat mobilization in cachectic cancer patients. Cachexia might improve if this lipolytic action of cancer cells could be halted. This study examined the lipolytic activities of media from four tumor cell lines after treatment with retinoic acid (RA), a cell differentiation inducer. An in vitro adipocyte bioassay measured lipolysis. All four tumor cell lines were intrinsically lipolytic, with elevated baseline lipolytic activities relative to fibroblast-conditioned controls (128% to 287% of control, p less than 0.05). After a 2-week exposure to RA in culture medium followed by 3 days of continued growth in fresh medium, two of four cell lines (both rat prostatic adenocarcinomas) showed significantly reduced lipolytic activities (16% and 61% of corresponding untreated controls, p less than 0.05). These reductions in lipolytic activity after RA treatment were not generalized phenomena; nor were they simply caused by cell differentiation, as the other cell lines (human malignant melanoma and human ovarian teratocarcinoma) showed no reductions despite evidence of cell differentiation. No effect on lipolytic activity was seen after only a 24-hour exposure to RA. We conclude that RA can affect the lipolytic activity of certain tumor cells in vitro, perhaps by influencing tumor-producing lipolytic factor(s).

Topics: Adenocarcinoma; Animals; Cell Line; Cell Transformation, Neoplastic; Female; Fibroblasts; Humans; Lipolysis; Male; Melanoma; Neoplasms; Rats; Teratoma; Time Factors; Tretinoin

D-alpha tocopheryl succinate (vitamin E succinate), which is known to induce differentiation and growth inhibition in murine B-16 melanoma cells, reduced basal and melanocyte-stimulating hormone (MSH)-stimulated adenylate cyclase (AC) activity in vitro. Vitamin E succinate treatment also reduced sodium fluoride- and forskoline-stimulated AC activity of melanoma cells in vitro. Treatment of cells with vitamin E succinate (6 micrograms/ml] for a period of 24 hours was sufficient to reduce MSH-stimulated AC activity. Other forms of vitamin E, such as d1-alpha tocopheryl nicotinate, d1-alpha tocopheryl acetate, and d1-alpha tocopherol, which did not affect growth or morphology of melanoma cells, were relatively less effective in altering basal and MSH-stimulated AC activity. Retinoic acid, which inhibited the growth of B-16 melanoma cells, also reduced basal and MSH-, NaF-, and forskolin-stimulated AC activity in vitro. Prostaglandin A2, which inhibited growth and altered morphology, did not change basal or MSH-stimulated AC activity. These results show that one of the mechanisms of action of vitamin E succinate and retinoic acid on melanoma cells may involve reduction of basal and MSH-sensitive AC activity, and this vitamin effect is not necessarily related to growth inhibition.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Colforsin; Melanocyte-Stimulating Hormones; Melanoma; Prostaglandins A; Sodium Fluoride; Tocopherols; Tretinoin; Tumor Cells, Cultured; Vitamin E

The ability of cationized ferritin (CF) to redistribute negatively charged cell surface molecules has been shown to increase after malignant transformation. Pretreatment of murine melanoma S91-C2 and B16-F1 cells with retinoic acid (RA), which suppresses their transformed phenotype, decreased the ability of CF to cluster surface anionic sites. In contrast, a similar pretreatment of RA-resistant mutant clone S91-C154 and subline B16-F10 caused only a minor reduction in CF-induced patching of anionic sites. These results indicate that the effect of RA on the redistribution of negatively charged cell surface molecules is related to the growth-inhibitory action of this vitamin A metabolite.

Topics: Animals; Cell Line; Ferritins; Melanoma; Sialoglycoproteins; Tretinoin

The functional properties of the melanoma-associated antigens detected by monoclonal antibodies (MAbs) AMF-6 and AMF-7 were investigated. These MAbs were selected previously because of their capacity to block the anti-melanoma reactivity of cytotoxic T-lymphocyte clones AMF-6 and AMF-7 detect a melanoma-associated proteoglycan (MW greater than 450-250 kDa) and a molecular complex, which under reducing conditions consists of 4 compounds of 120, 95, 29 and 25 kDa respectively. AMF-6 reacted strongly with all 30 cultured melanomas and all 41 melanomas in frozen tissue sections. Significant cross-reactivity was only observed with nevi and perineurium, whereas normal skin melanocytes were negative. AMF-7 reacted with all 25 cultured melanomas and all 34 melanomas in frozen sections. AMF-7 cross-reacted with a proportion of nevi and endothelial cells from small vessels. The antigen detected by AMF-6 and AMF-7 could not be modulated by retinoic acid or recombinant gamma-IFN, which induced or enhanced the expression of HLA-DR, HLA-DQ and Class-I MHC antigens. In addition, the antigens were not readily modulated when cells were incubated in excess amounts of AMF-6 and AMF-7. Interestingly, the antigen detected by AMF-7 was strongly associated with the adhesion and cytoplasmic spreading of melanoma cells to plastic surfaces and monolayers of vascular endothelial cells. AMF-6 did not block the adhesion of melanoma cells but delayed cytoplasmic spreading. Both AMF-6 and AMF-7 blocked fibronectin-induced chemotaxic motility and chemokinesis of melanoma cells. In addition to their membrane localization, the antigens detected by AMF-6 and AMF-7 were also abundant in extracellular adhesion plaques deposited by cultured melanoma cells. Our results indicate that the high-MW melanoma-associated proteoglycan and the antigen detected by AMF-7 are associated with adhesion and/or cytoplasmic spreading and motility of human melanoma cells, suggesting that these antigens are associated with the (hematogenic) dissemination of human melanoma. This is supported by the finding that AMF-7 stained primary tumors heterogeneously, whereas metastases were homogeneously stained.

Topics: Animals; Antibodies, Monoclonal; Antigens, Surface; Cell Adhesion; Cell Line; Cell Movement; Epitopes; Fluorescent Antibody Technique; Histocytochemistry; Humans; Interferon-gamma; Melanoma; Mice; Mice, Inbred BALB C; Plastics; Recombinant Proteins; Tretinoin

The effects of retinol, all-trans-retinoic acid, isotretinoin and etretinate on the activity of basement membrane collagen degrading enzyme was studied in melanoma cells. The results indicated that retinoids at concentrations of up to 10(-6) M did not significantly affect type IV collagenolytic activity in these cells in vitro. Since type IV collagenolytic enzyme may be involved in the metastatic potential of tumour cells, it appears that retinoids do not affect the metastatic potential of melanoma cells by affecting type IV collagenolytic activity.

Topics: Cells, Cultured; Etretinate; Humans; Isotretinoin; Melanoma; Microbial Collagenase; Neoplasm Metastasis; Retinoids; Skin Neoplasms; Tretinoin; Vitamin A

Vitamin A inhibits growth and increases the activity of cAMP-dependent protein kinase in B16 mouse melanoma cells. In this report we show that retinoic acid (RA) treatment of intact cells alters their subsequent in vitro protein phosphorylation, but we could not demonstrate any changes in in vivo protein phosphorylation. A 48-h treatment with RA results in a concentration-dependent decrease of protein phosphorylation of a 95K molecular weight (MW) protein in both supernatant and particulate fractions. The phosphorylation of this protein does not appear to be regulated by cAMP. Proteins at 92K and 82K MW in the supernatant fraction are increased in phosphorylation. The former (but not the latter) is regulated by cAMP. In the particulate fraction a variety of proteins 12K-68K MW are increased in phosphorylation, as the cells are treated with increasing amounts of RA. The phosphorylation of most of these proteins is regulated by cAMP. Another inhibitor of B16 cell growth, melanocyte-stimulating hormone (MSH) also alters protein phosphorylation. At short incubation periods (1 h), this hormone stimulates phosphorylation of a number of proteins (17-40K MW), while in longer incubation periods (48 h) phosphorylation is inhibited. All of these phosphorylations appear to be regulated by cAMP. We attempted to repeat these observations using intact-cell phosphorylation with 32PO4. In two experiments we saw small changes in the phosphorylation of proteins. In most experiments, however, we could find no change in the phosphoproteins. Further experiments have led us to question the in vivo phosphorylation, since treatment of the cells with MSH, cholera toxin, or db-cAMP also did not affect intact-cell protein phosphorylation. We have previously documented that under these latter conditions cAMP levels are greatly elevated and cAMP-dependent protein kinase is activated. The in vitro phosphorylation results suggests that in RA-treated cells, kinase activities and/or protein substrate levels are changing. However, the physiological significance of the particular MW phosphoproteins changes we have described must await resolution of the in vivo phosphorylation data.

Topics: Animals; Cell Division; Cyclic AMP; Enzyme Activation; Melanocyte-Stimulating Hormones; Melanoma; Mice; Neoplasm Proteins; Phosphorylation; Protein Kinases; Tretinoin

Five human melanoma cell lines (C8146C, C8161, C82-7A, C83-2CY and MIRW5) were shown to contain a significant number of melanoma colony-forming units resistant to single-agent treatment by dexamethasone, alpha-interferon and trans-retinoic acid. These biological modifiers were combined with difluoromethylornithine into a low-dose combination using concentrations below pharmacologically achievable levels. The suppression of melanoma colony formation induced by this combination was consistent and significantly higher than that seen with any single agent, colony formation being reduced by an average of 90%. Leaving either DEX or DFMO out of the 4-agent combination resulted in a significant decrease in the observed inhibition. This was also verified by the addition of putrescine which inhibited only the DFMO activity. Median effect analysis of the DFMO + IFN inhibition of C8161 cells demonstrated that the 2 agents interacted synergistically over the entire dose-response curve. Of the high-dose combination-treated melanoma colony-forming units, 97% did not form small growth units; most remained as arrested single cells, but the cells and small growth units could still metabolize tetrazolium stain after the experiment, suggesting that the high-dose combination arrested the growth of the melanoma colony-forming units via a non-cytotoxic mechanism.

Topics: Cell Line; Cell Survival; Dexamethasone; Drug Combinations; Drug Synergism; Eflornithine; Humans; Interferon Type I; Melanoma; Ornithine; Putrescine; Tretinoin

Melanin synthesis of B16 mouse melanoma cells was found to be stimulated dose and time dependently by 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], the hormonal form of vitamin D3. The stimulation of melanogenesis resulted from an increase in the activity of tyrosinase, a key enzyme in melanin synthesis. The minimum dose required for this stimulation was as low as 0.05 ng/ml, or 0.12 nM, a physiological level of plasma 1 alpha,25(OH)2D3. The stimulation by 1 alpha,25(OH)2D3 was specific; other derivatives of vitamin D3 caused no stimulation at a concentration of 500 ng/ml. When the cells were plated on agar plates, the proportion of dark or black colonies was not increased by the exposure to 1 alpha,25(OH)2D3. Furthermore, this compound did not induce melanin synthesis of an amelanotic variant. Thus, its stimulatory effect seemed to be due to stimulation of melanin synthesis of melanotic cells, rather than to conversion of amelanotic clones to melanotic ones. 1 alpha,25(OH)2D3 did not induce intracellular cyclic adenosine 3':5'-monophosphate, while cholera toxin induced cyclic adenosine 3':5'-monophosphate and stimulated melanin synthesis and tyrosinase activity much more than did 1 alpha,25(OH)2D3, suggesting that 1 alpha,25(OH)2D3 stimulates melanin synthesis by a cyclic adenosine 3':5'-monophosphate-independent mechanism. B16 melanoma cells contained specific receptors for 1 alpha,25(OH)2D3. Scatchard plot analysis revealed two types of receptor; the high-affinity receptor had a Kd of 18.3 pM and an Nmax of 10.6 fmol/mg of protein. The specificity of receptor binding was demonstrated by studies showing that, for 50% displacement of 1 alpha,alpha,25(OH)2D3 binding, other derivatives were required at 500 times higher concentrations or more. In contrast to 1 alpha,25(OH)2D3, retinoic acid inhibited melanin synthesis and tyrosinase activity of B16 melanoma cells dose and time dependently. On simultaneous treatment, 1 alpha,25(OH)2D3 and retinoic acid caused mutual interference, and a balance between their respective stimulating and inhibitory effects was obtained at a molar ratio of 10:1; i.e., with 10 nM 1 alpha,25(OH)2D3 and 1 nM retinoic acid.

Topics: Animals; Calcitriol; Cells, Cultured; Cyclic AMP; Melanins; Melanoma; Mice; Receptors, Calcitriol; Receptors, Steroid; Tretinoin

To a limited extent, we corroborated a previous report of human neuroblastoma sensitivity to 13-cis-retinoic acid. Seven cultured human neuroblastoma, two primitive neuroectodermal tumor, and one melanoma cell line were exposed to 0.001 to 10.0 microM 13-cis-retinoic acid for six to fourteen days. The neuroblastoma cell line, SK-N-DZ, was the only cell line lysed by all concentrations of 13-cis-retinoic acid. The other cell lines were refractory to concentrations as high as 10 microM. Increased cell process formation was observed in three neuroblastoma, SK-N-SH, SK-N-BE, SK-N-LE, and one melanoma cell line. We conclude that sensitivity to 13-cis-retinoic acid is unevenly distributed among histogenetically similar tumors from different patients.

Topics: Cell Line; Cell Survival; Humans; Melanoma; Neoplasms, Germ Cell and Embryonal; Neuroblastoma; Tretinoin; Tumor Stem Cell Assay

Topics: Animals; Cell Division; Cell Line; Etretinate; Kinetics; Melanoma; Mice; Microscopy, Electron, Scanning; Tretinoin

Growth cessation and cell death of exponentially proliferating Harding-Passey melanoma cells (HPM-73 line) in monolayer culture resulted in the presence of 3.3 X 10(-5) M retinal, while retinol and retinoic acid caused growth retardation at 3.3 X 10(-5) M. Also at 1 X 10(-5) M, the growth-inhibitory effect of retinal was more pronounced than that of retinol or retinoic acid. Following serum removal from the culture media, all 3 retinoids at 3.3 X 10(-6) M or 1 X 10(-5) M revealed cytotoxic effects within 3 days as demonstrated by cell loss from the substratum. Thus, the presence of serum has "protective" effects. Addition of retinal, retinoic acid or retinol at 1 X 10(-5) M to cultures in stationary growth phase did not result in cell loss during period of 6 days. C57Bl mice with B16 melanotic melanoma were i.p. injected during 10 days with retinoids (30 or 100 mcg per mouse daily). All retinoids inhibited B16 tumor growth in vivo. In this respect, retinoic acid was the most effective one. The cellular melanin content of cultured HPM-cells and of B16 melanotic melanoma in vivo was elevated after treatment with retinoids; retinal having the strongest effect.

Topics: Animals; Cell Line; In Vitro Techniques; Melanins; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Retinaldehyde; Retinoids; Stimulation, Chemical; Tretinoin; Vitamin A

Previous studies have demonstrated that retinoids possess antineoplastic properties against melanoma. The purpose of this study was to determine whether topically applied retinoic acid could prevent melanoma development in syngeneic mice after intracutaneous cell inoculation. Trans-retinoic acid in DMSO was applied daily for 28 days after melanoma implantation and tumor growth was quantitated by the uptake of [14C]thiouracil, a tracer compound specific for melanoma which is incorporated linearly according to the weight of the tumor. Marked reduction in tumor growth was noted at the highest concentration (0.1%) tested and lesser but significantly decreased tumor growth patterns were also realized at lower concentrations in a dose-dependent manner. Thus, topically applied retinoic acid is capable of inhibiting S91 melanoma growth in vivo.

Topics: Administration, Topical; Animals; Carbon Radioisotopes; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Male; Melanoma; Mice; Mice, Inbred DBA; Neoplasm Transplantation; Skin Neoplasms; Thiouracil; Tretinoin

Retinoic acid inhibits the proliferation of the murine melanoma clone S91-C-2 cells, enhances the glycosylation of specific cell surface sialoglycoproteins, and stimulates sialytransferase activity. Mutant clones, selected from the S91-C-2 cells for resistance to the growth-inhibitory effect of retinoic acid, were used to explore whether cell surface modulation by retinoic acid is related to growth inhibition. Glycoprotein synthesis was assessed by analysis of [3H]glucosamine incorporation into glycoconjugates, and cell surface sialo- and galactoglycoproteins were analyzed after radiolabeling by the NaIO4:NaB3H4 and the neuraminidase plus galactose oxidase:NaB3H4 methods, respectively. The cells were solubilized and the labeled molecules were separated by polyacrylamide gel electrophoresis and identified by fluorography. Sialytransferase activity was measured in detergent-solubilized cells, using cytidine 5' -monophosphate-[14C]sialic acid as a sugar donor and asialofetuin as an exogenous acceptor. The results demonstrated that retinoic acid enhanced [3H]glucosamine incorporation into a Mr 160,000 glycoprotein in the S91-C-2 cells but not in any of the resistant mutant clones, while the pattern of [35S]methionine-labeled proteins was not modified in either the sensitive or the resistant clones. Radiolabeling of a Mr 160,000 sialoglycoprotein on the surface of S91-C-2 and of several retinoic acid-sensitive subclones of S91-C-2 was augmented by retinoic acid. A considerably smaller effect was observed on the labeling of Mr 160,000 sialoglycoprotein on one of the resistant clones, and no significant effect could be detected on the other resistant mutant clones. Sialytransferase activity was increased 2- to 3-fold by retinoic acid in the S91-C-2 cells and in several sensitive subclones, but not in any of the resistant mutant clones. Tetradecanoylphorbol acetate, which inhibits the proliferation of both retinoic acid-sensitive and retinoic acid-resistant cells, failed to increase either sialyltransferase activity or cell surface labeling of sialoglycoproteins. These findings suggest that the ability of retinoic acid to stimulate sialyltransferase activity and glycosylation of cell surface glycoproteins is related to the growth-inhibitory effect of this compound.

Topics: Animals; Cell Division; Cell Line; Cell Membrane; Clone Cells; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Glycoproteins; Melanoma; Mice; Mutation; Sialoglycoproteins; Sialyltransferases; Transferases; Tretinoin

We have previously reported [(1980) J. Biol. Chem. 255, 5999-6002] that retinoic acid inhibited growth and increased cyclic-AMP-dependent protein kinase activity in mouse melanoma cells. A variant melanoma line having depressed levels of cyclic-AMP-dependent protein kinase was not growth-inhibited by retinoic acid. In this report we describe the effect of retinoic acid on cyclic AMP binding proteins in B16 mouse melanoma cells. Using the technique of photoaffinity labeling, we found three major proteins of Mr 49 000, 52 000, and 55 000 which were specifically labeled with 8-N3-[32P]AMP in both control and treated cells. Based upon their molecular weight, relative affinity for 8-N3-[32P]AMP and comigration with standards, we have designated the 49 000-Mr and 55 000-Mr species as RI and RII respectively. The position of the intermediate band (Mr 52 000) was not affected by pre-incubation with ATP or alkaline phosphatase, and two-dimensional gel analysis indicated that it had the same pI as RI. Retinoic acid increased the 8-N3-[32P]AMP labeling of RI within 24 h, reaching a maximal six fold increase by 48 h. These increases were limited to the 40 000 X g supernatant fraction and occurred prior to any growth inhibition. By using increasing concentrations of 8-N3-cAMP we were able to construct a saturation curve for RI binding. Calculation of apparent Kd values from these curves showed nearly identical affinities for RI binding of 8-N3-cAMP from control and retinoic-acid-treated cells. Therefore we conclude that retinoic acid is increasing the amount of RI rather than altering its properties. Corroboration of these results was obtained by DEAE-cellulose chromatography. Peak I (corresponding to type I protein kinase) from retinoid-treated cells was increased about six fold in binding activity.

Topics: Affinity Labels; Animals; Carrier Proteins; Chromatography, DEAE-Cellulose; Cyclic AMP Receptor Protein; Electrophoresis, Polyacrylamide Gel; Melanoma; Mice; Neoplasm Proteins; Photochemistry; Tretinoin

The cloning efficiencies of a murine melanoma cell line (S91 CCL 53.1) and a human melanoma cell strain (C8146c) were inhibited by dexamethasone (DEX), prostaglandin A1 (PGA1), and beta-all-trans-retinoic acid (RA) in a dose-dependent manner. Murine melanoma tumor colony-forming units (MTCFU) were inhibited more than 99% by DEX (1 X 10(-7) M) and RA (1 X 10(-7) M) with a concentration needed to produce a 50% reduction in colony formation for both hormones of 5 X 10(-9) M. Combinations of DEX and RA effected a synergistic inhibition on colony formation, which was reflected by a 11/2 log reduction in the hormone concentration needed to produce a greater than 99% inhibition of colony formation. When PGA1 was added to DEX and RA, a greater than additive reduction in colony formation was observed. Human MTCFU from cell strain C8146c were inhibited more than 85% at an RA concentration of 1 X 10(-7) M, but they were reduced only to 40% of control at a DEX concentration of 1 X 10(-6) M. DEX-RA produced an additive inhibition of colony formation. Addition of submaximal amounts of PGA1 to DEX-RA combinations or to either hormone alone resulted in synergistic reduction of human MTCFU. These results demonstrated that the proliferative potential of human and murine melanomas can be simultaneously regulated by DEX, PGA1, and RA.

Topics: Agar; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Division; Cell Line; Dexamethasone; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Humans; Melanoma; Mice; Prostaglandins A; Tretinoin

Necessary for growth and differentiation in many normal tissues and capable of inducing differentiation in human promyelocytic cell lines, retinoids were the subject of this study. Specifically, effects of 13-cis-retinoic acid and 13-trans-retinoic acid on the growth of normal human bone marrow cells in soft-agar system were studied. Both short-term incubation and continuous exposure to retinoic acid caused a decreased number of granulocyte colonies and an increased cluster-to-colony ratio. This effect was concentration-dependent. Examination of specimens stained with Wright-Giemsa or nitro blue tetrazolium stains showed a progressive increase in the percentage of immature granulocytic precursors with increasing concentrations of retinoic acid. No effect of retinoic acid was seen on a number of human tumor cell lines. Retinoic acid blocked both differentiation and proliferation and appeared to do so by specific, noncytotoxic mechanisms in normal human bone marrow cells.

Topics: Bone Marrow; Bone Marrow Cells; Burkitt Lymphoma; Cell Division; Cell Line; Clone Cells; Colony-Forming Units Assay; Colony-Stimulating Factors; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Humans; Leukemia, Myeloid; Melanoma; Multiple Myeloma; Osteosarcoma; Tretinoin

Retinoic acid (RA) treatment of murine S91-C2 melanoma cells has been found to augment the activity of glycoprotein: sialyltransferase in a dose-dependent and time-dependent process. The enzymatic activity in cells treated with 10 microM RA reached a maximal level, 3-fold higher than in untreated cells, 72 h after initiation of treatment. In contrast, the addition of RA directly into the reaction mixture had no stimulatory effect on sialyltransferase. The endogenous glycoproteins to which sialic acid is transferred from cytidine monophosphate (CMP)-[14C] sialic acid by the action of sialyltransferase have been identified by fluorography after polyacrylamide gel electrophoresis. One of these acceptors, a glycoprotein of Mr 160 000, comigrated in gel electrophoresis with a cell surface sialoglycoprotein that can be labeled by the periodate-tritiated borohydrate procedure more intensely on intact RA-treated than on untreated cells. Removal of sialic acid residues exposed on the surface of either control or RA-treated cells enhanced 2- to 3-fold the transfer of sialic acid to endogenous acceptors. These results suggest that the increased sialyltransferase activity in RA-treated melanoma cells may be responsible for the enhanced sialylation of certain cell surface glycoproteins. RA treatment of several other tumor cell lines also resulted in stimulation of sialyltransferase activity indicating that this effect of RA is not limited to the S91-C2 melanoma cells.

Topics: Animals; beta-D-Galactoside alpha 2-6-Sialyltransferase; Cell Line; Clone Cells; Dose-Response Relationship, Drug; Glycoproteins; Melanoma; Membrane Proteins; Mice; N-Acetylneuraminic Acid; Sialic Acids; Sialyltransferases; Transferases; Tretinoin

Topics: Animals; Cell Membrane; Dose-Response Relationship, Drug; Glycolipids; Glycoproteins; Melanoma; Mice; Molecular Weight; Time Factors; Tretinoin

Human malignant melanoma cell lines have been divided into three broad groups on the basis of morphology, pigmentation, tyrosinase levels, the 2-dimensional electrophoretic patterns of their [3H]glucosamine-labeled glycoproteins and the presence or absence of an extracellular matrix of fibronectin. The most pigmented cell lines were characterized by the synthesis of a novel glycoprotein with a molecular weight of 75,000 and the absence of a fibronectin matrix. As cultured skin melanocytes also had these characteristics, this group of melanomas appears to be the most differentiated. Melanoma cell lines in the amelanotic group were characterized by the synthesis of high levels of HLA-DR antigen and by the production of an extracellular fibronectin matrix.

Topics: Cell Division; Cell Line; Electrophoresis, Polyacrylamide Gel; Fibronectins; Glycoproteins; Humans; Melanocytes; Melanoma; Molecular Weight; Tretinoin

Retinoic acid inhibits both the anchorage-dependent and the anchorage-independent growth of the murine melanoma S91-C-2 cells. To explore the mechanism of these effects, several mutant cell clones resistant to retinoic acid-induced growth inhibition have been derived from the S91-C-2 cells by exposing them to the mutagen ethyl methane sulfonate and plating in soft agarose in the presence of 1 microM beta-all-trans-retinoic acid. Under such conditions, the nonmutagenized S91-C-2 cells failed to grow; however, 2 X 10(-6) of the mutagenized cells did form colonies. These colonies were isolated, expanded in culture, and recloned in agarose containing retinoic acid. Five cell clones that retained their drug-resistant phenotype after repeated subculture for 3 months, in the absence of retinoic acid, were characterized further. They were found to be 3- to greater than 1000-fold and 100- to greater than 100-fold resistant to retinoic acid-induced inhibition of anchorage-independent and anchorage-dependent growth relative to the wild-type C-2 cells, respectively. The rate of uptake of [3H]-retinoic acid by the resistant cell clones was similar to that of the sensitive C-2 cells, indicating that resistance is not the result of reduced uptake. Analysis of cytoplasmic retinoic acid-binding protein revealed that it is present in the most resistant clones in amounts that are similar to or even greater than those found in the sensitive S91-C-2 cells. These results indicate that resistance is not the result of the absence of the binding protein. The retinoic acid-resistant mutants exhibited cross-resistance to related retinoids such as 13-cis-retinoic acid and all-trans-retinol as well as to the arotinoid p-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl) propenyl]benzoic acid suggesting that they all share a similar mechanism of action. These resistant mutants may provide a useful system for further studies of the molecular processes through which retinoic acid exerts its antiproliferative effects.

Topics: Animals; Cell Division; Clone Cells; Drug Resistance; Karyotyping; Melanoma; Mice; Mutation; Tretinoin

The incorporation of 2-[35S]thiouracil and two of its derivatives into murine melanomas, in vivo, was studied. It was confirmed [J. R. Whittaker, J. Biol. Chem., 246, 6217--6226 (1971)] that 2-thiouracil has a marked affinity for melanin-producing tissue and that an affinity for such tissue could be sustained by 5-substituted 2-thiouracils. A series of derivatives of arotinoids and retinoids, with or without a 2-thiouracil group as a potential carrier to obtain affinity for melanomas, was examined for cytostatic activity, in vitro. None of these showed significant activity against murine melanomas.

Topics: Animals; Antineoplastic Agents; Cells, Cultured; Chemical Phenomena; Chemistry; Melanoma; Mice; Mice, Inbred DBA; Neoplasms, Experimental; Thiouracil; Tissue Distribution; Tretinoin

Transglutaminase (TGase; R-glutaminyl-peptide:amine gamma-glutamyltransferase, EC 2.3.2.13) and ornithine decarboxylase (ODCase; L-ornithine carboxy-lyase, EC 4.1.1.17) activities were measured after the addition of retinoid analogs to Chinese hamster ovary (CHO) cells released from quiescence and Cloudman S91 (CCL 53.1) mouse melanoma cells stimulated to differentiate with alpha-melanocyte-stimulating hormone (MSH, melanotropin). In both cell culture lines, we detected a biphasic increase in TGase activity and a single peak of ODCase activity within 7 hr after release or stimulation. Retinoid analogs altered the expression of the initial TGase peak in both CHO and melanoma cells. Retinol increased the activity of TGase 1 hr after release in CHO cells, and the activity remained elevated until hr 4. A broad peak of TGase activity also occurred after the addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODCase, and after addition of alpha-difluoromethylornithine plus retinol. In mouse melanoma cells, retinoic acid plus MSH markedly enhanced the activity of the initial TGase peak compared to MSH alone. Retinoic acid alone also increased TGase activity biphasically in these cells without the addition of MSH. These studies suggest that retinoid effects that increase TGase activity may alter the ODCase expression in proliferation and differentiation.

Topics: Animals; Carboxy-Lyases; Cell Differentiation; Cell Line; Cricetinae; Cricetulus; Cycloheximide; Dactinomycin; Female; gamma-Glutamyltransferase; Kinetics; Melanocyte-Stimulating Hormones; Melanoma; Mice; Neoplasms, Experimental; Ornithine Decarboxylase Inhibitors; Ovary; Tretinoin; Vitamin A

Two melanoma cell lines, each derived from a different patient with metastatic disease, were very similar in their appearance, their growth characteristics, and their tendency to differentiate and to pigment in culture as they become confluent. These lines, UCT-Mel 1 and UCT-Mel 2, were used to study the effects of retinoic acid and other derivatives of vitamin A. When added to UCT-Mel 1 cells, retinoids had only a modest effect on plasminogen activator release and were without measurable effect on morphology, growth, or tyrosinase synthesis. In contrast, when added to UCT-Mel 2 cells, retinoids appeared to induce a more differentiated state evident as an inhibition of cell proliferation and the assumption of a dendritic morphology. Paradoxically, however, retinoids caused a striking inhibition of the density-dependent intracellular accumulation of tyrosinase and melanin that was taken to represent spontaneous in vitro differentiation. Culture of UCT-Mel 2 cells in the presence of retinoic acid resulted in initial inhibition followed by marked stimulation of cellular plasminogen activator release. The data suggest that the manner in which retinoids exert their effects on cells in vitro does not depend on the histological origin of the tumor cells being studied but on the innate responsiveness of that particular cell line to the retinoid or compound in question.

Topics: Aged; Cell Division; Cell Line; Female; Humans; Kinetics; L-Lactate Dehydrogenase; Melanins; Melanoma; Monophenol Monooxygenase; Plasminogen Activators; Tretinoin

Investigation of retinoids for anticancer activity in humans, either in the chemopreventive or treatment mode, has been little studied. We summarize here our ongoing investigations in four different areas: (1) secondary prevention of cervical dysplasia with topical application of all-trans-retinoic acid; (2) adjuvant treatment of resected high-risk stage I and II malignant melanoma with bacille Calmette Guérin (BCG) plus or minus oral vitamin A; (3) topical vitamin A acid therapy for cutaneous metastatic melanoma; an (4) oral isotretinoin as an anticancer agent.

Topics: Diterpenes; Female; Humans; Isomerism; Isotretinoin; Melanoma; Neoplasms; Palmitates; Retinyl Esters; Skin Neoplasms; Tretinoin; Uterine Cervical Dysplasia; Vitamin A

Topics: Animals; Cell Adhesion; Cell Division; Cell Line; Clone Cells; Female; Fibrinolysis; Fibrosarcoma; HeLa Cells; Humans; Melanoma; Mice; Neoplasms; Osteosarcoma; Plasminogen Activators; Tretinoin; Vulvar Neoplasms

Topics: Antineoplastic Agents; Brain Neoplasms; Etretinate; Female; Humans; Male; Melanoma; Ovarian Neoplasms; Teratoma; Testicular Neoplasms; Tretinoin

Topics: Daunorubicin; Dicarboxylic Acids; Drug Combinations; Drug Resistance; Humans; Levodopa; Melanocyte-Stimulating Hormones; Melanoma; Methotrexate; Nitrosourea Compounds; Receptors, Estrogen; Tretinoin

It has been reported previously that malignancy in hybrid cells is associated with changes in two glycoproteins of molecular mass 90,000 and 100,000. In the current paper the characteristics of a clonal derivation of a murine melanoma cell line (PG19G-), selected for growth in low levels of glucose, are described. The cells had considerably reduced amounts of the 90 K and 100 K glycoproteins as judged by lectin affinity-labelling. When the level of glucose in the medium is raised the two glycoproteins become detectable after a few hours. This inducibility is specific for D-glucose and D-mannose at concentrations in excess of 300 mg/l and is inhibited by cytochalasin B but not by cytochalasin A. The 90 K glycoprotein differs from the 100 K in that its level varies with the glucose concentration even in the parent cell line PG19 and in other tumour lines. This is not the case for the 100 K glycoprotein, the synthesis and/or glycosylation of these two glycoproteins are, therefore, governed by separate control mechanisms. The relationship of the glucose-sensitive proteins to the glucose transporter is discussed.

Topics: Animals; Cell Line; Glucose; Glycoproteins; Melanoma; Mice; Mice, Inbred C57BL; Molecular Weight; Neoplasm Proteins; Neoplasms, Experimental; Tretinoin

Retinoic acid reduces the growth rate of mouse S91 melanoma cells in culture and increases the proportion of cells in the G1 phase of the cell cycle. Because of the integral role protein synthesis has been shown to play in growth control we studied the effect of retinoic acid on the protein synthesis machinery with a cell-free system developed from the melanoma cells. This system was capable of translating endogenous mRNA, exogenous globin mRNA, and the synthetic template poly(U). Of the above activities of the protein synthesis system only the translation of endogenous mRNA was reduced significantly in the cell-free system prepared from retinoic acid-treated cells. Analyses of the amount and function of RNA revealed that treatment with retinoic acid leads to reductions in total RNA content, in the proportion of ribosomes in polysomes, in the amount of poly(A)RNA, and in the amount of polysome-associated mRNA. All these effects of retinoic acid contribute to the decrease in protein synthesis activity of treated cells. Two-dimensional electrophoresis analysis of L-[35S]methionine-labeled proteins produced by untreated and treated cells revealed only a few quantitative differences. We suggest that retinoic acid-induced suppression of protein synthesis activity may be the cause for growth inhibition.

Topics: Animals; Cell Division; Cell Line; Cell-Free System; Isoelectric Point; Melanoma; Mice; Molecular Weight; Neoplasm Proteins; Neoplasms, Experimental; Polyribosomes; RNA, Messenger; Tretinoin

Retinoic acid (RA), which reduces the rate of cell proliferation in S91 mouse melanoma clone C2 cells, was found to stimulate the expression of their melanotic phenotype. RA treatment also induced the extension of long cellular processes. The RA effects on melanogenesis included stimulation of tyrosinase activity and augmentation of cellular melanin content to levels 3- to 4-fold higher than in untreated cultures at similar cell densities. These effects became apparent after 48 hours of exposure to 10(-5) M RA and increased thereafter. Half-maximal stimulation in cells treated for 6 days occurred at 5 X 10(-7) M RA. Although the degrees of melanogenesis enhancement by RA (10(-5) M) and by alpha-melanocyte stimulatory hormone (2 X 10(-7) M) were similar, the former did not alter the intracellular cAMP level, whereas the latter induced a transient 4-fold increase. In high-passage (p28) cells, as well as in low-passage cells (less than p10) treated with tyrosinase inhibitor phenylthiocarbamate, melanin synthesis was suppressed in the absence and presence of RA, yet the ability of RA to inhibit cell proliferation was not compromised. In the presence of the tumor promotor phorbol myristate acetate (greater than 5 X 10(-9) M) melanin synthesis in control as well as in cells exposed to RA was dramatically inhibited. Phorbol which is not active in tumor promotion had no effect on melanogenesis. In addition to RA, other retinoids, such as 13-cis-retinoic acid, retinyl acetate, the TMMP analog of RA and the phenyl analog of RA, but not the pyridyl analog of RA or retinyl palmitate, also inhibited cell growth and enhanced melanin synthesis.

Topics: Animals; Cell Division; Cells, Cultured; Cyclic AMP; Enzyme Activation; Melanins; Melanocyte-Stimulating Hormones; Melanoma; Mice; Monophenol Monooxygenase; Neoplasms, Experimental; Tetradecanoylphorbol Acetate; Tretinoin

We employ the murine S91 and the human Hs939 melanoma cell lines for the characterization of various biochemical changes induced by retinoids. Retinoic acid (RA) causes a time-dependent, and reversible reduction in cell proliferation rate in liquid medium and inhibits growth in agar. The proportion of cells in the G1 phase of the cell cycle increases in RA-treated cells, and the uptake of TdR, UdR and Leu decreases. The growth inhibitory effect of RA is apparently not mediated via labilization of lysosomes, increase in cAMP or changes in the synthesis of prostaglandins or polyamines. Exposure to RA stimulates tyrosinase activity and increases melanin content severalfold over the levels found in untreated cells. Various retinoids exhibit the activities of RA; however, their potencies vary depending on their structure. Those possessing a free -COOH at C-15 are usually more effective than those with a different group or with a derivatized carboxyl. A positive correlation exists between the ability of retinoids with a free -COOH in C-15 to inhibit growth and to bind to an RA-binding protein found in the S91 melanoma cells. Future studies will explore recently discovered changes in the glycosylation of cell surface components and their relationship to the phenomena described here.

Topics: Animals; Cell Cycle; Cell Differentiation; Cell Division; Cell Line; DNA, Neoplasm; Humans; Kinetics; Melanoma; Mice; Mice, Inbred Strains; Neoplasms, Experimental; Tretinoin

Topics: Animals; Cell Cycle; Cell Division; Glycosaminoglycans; Kinetics; Melanoma; Mice; Neoplasms, Experimental; Tretinoin

Topics: Cell Division; Female; Humans; Melanoma; Ovarian Neoplasms; Structure-Activity Relationship; Tretinoin; Urinary Bladder Neoplasms

Murine B16 melanoma sublines have been cloned or selected in vivo for preference of bloodborne metastatic colonization of lung, ovary, or brain. These sublines show differing metastatic properties and cell surface alterations that correlate with a preference for metastatic colonization sites. When the sensitivities of these selected sublines to certain drugs (beta-all-trans retinoic acid or 1,3-bis (2-chlorethyl)-1-nitrosourea (BCNU) were examined, the in vivo-selected sublines were more resistant to growth inhibition in vitro by cytostatic (retinoic acid) or cytotoxic (BCNU) drugs than was the parental B16 line.

Topics: Animals; Carmustine; Cell Line; Clone Cells; Drug Evaluation, Preclinical; Melanoma; Mice; Neoplasms, Experimental; Tretinoin

Two patients with cutaneous metastatic melanoma were treated with a topical retinoid, beta-all-trans-retinoic acid. Complete regression of the treated lesions was noted in one patient and a partial response was seen in the other patient. The mechanism of anti-tumour action of the retinoids is not completely known but binding to intracytoplasmic receptors with promotion of cellular differentiation, alteration of membranes, and immunological adjuvant effects may be involved.

Topics: Administration, Topical; Adult; Female; Humans; Male; Melanoma; Middle Aged; Neoplasm Metastasis; Skin Neoplasms; Tretinoin

The influence of all trans-retinoic acid on cyclic AMP metabolism was examined in B16-F1 mouse melanoma cells. Treatment of these cells with retinoic acid resulted in a dose-dependent inhibition of cell growth which was accompanied by a concentration-dependent increase in both basal and cyclic AMP-stimulated protein kinase activity, Intracellular levels of cyclic AMP, however, were not altered by retinoid treatment. A protein kinase-deficient variant of B16-F1 (MR-4) did not exhibit decreased growth or increased protein kinase activity in response to retinoic acid treatment. At least 24 h of incubation was required before increased protein kinase activity could be detected in treated B16-F1 cells. Retinoic acid treatment increased the Vmax of protein kinase, but the Ka for cyclic AMP activation was not altered. These findings suggest that in B16 mouse melanoma cells, cyclic AMP-dependent protein kinase may be a target for the growth inhibitory effects of the retinoid.

Topics: Animals; Cell Division; Cell Line; Cyclic AMP; Enzyme Activation; Melanoma; Mice; Neoplasms, Experimental; Protein Kinases; Tretinoin

Retinoic acid was found to be a potent stimulant of pigmentation in human Hs939 melanoma cells. Exposure to 1 microM retinoic acid for longer than four days caused both a decrease in the rate of cell proliferation and a concomitant increase in melanogenesis. These effects of retinoic acid progressed lin-early in a time-dependent and a dose-dependent fashion such that at the end of a seven-day treatment cell growth was inhibited by approximately 65%, and both melanin content and tyrosinase activity increased more than three-fold over the control. Interpolation of the dose-response curves indicated that 3 nM retinoic acid would cause half-maximal melanogenesis stimulation. No elevation in the level of cyclic adenosine 3':5'-monophosphate could be detected in the melanoma cells following various periods of exposure to retinoic acid, and the cells were unresponsive to alpha-melanocyte-stimulating hormone. In the presence of the tyrosinase inhibitor phenylthiocarbamate, retinoic acid was capable of inhibiting cell proliferation without enhancing melanin synthesis. The tumor promoter phorbol myristate acetate did not affect either the proliferation or the differentiation of the Hs939 melanoma cells. However, the enhancement of melanogenesis by 1 microM retinoic acid was inhibited by 66% in the presence of 0.1 microM phorbol myristate acetate. The tumor promoter did not reverse the growth-inhibitory effect of retinoic acid. Phorbol, a non-tumor promoter, was effective. Other retinoids, such as 13-cis-retinoic acid, retinyl acetate, nd the trimethylmethoxyphenyl analog of retinoic acid, also inhibited the proliferation and enhanced melanin production in the Hs939 cells. In contrast, retinyl palmitate, the phenyl analog of retinoic acid, and the pyridyl analog of retinoic acid were ineffective.

Topics: Cell Division; Cell Line; Cyclic AMP; Dose-Response Relationship, Drug; Humans; Melanins; Melanocyte-Stimulating Hormones; Melanoma; Monophenol Monooxygenase; Neoplasms, Experimental; Phorbol Esters; Stimulation, Chemical; Thiocarbamates; Tretinoin

The effect of four different retinoids [retinol, 13-cis-retinoic acid (Ro4-3780), beta-all-trans-retinoic acid, and aromatic retinoic acid ethyl ester (Ro10-9359)] on the cellular proliferation (cell number) and biochemical differentiation (tyrosinase activity) of a human melanoma cell line (MIRW) and three subclones was assessed. All four retinoids (10(-6) M) inhibited the cellular proliferation (36 to 42%) and stimulated tyrosinase activity (58 to 72%) in the parent cell line to a similar extent. In contrast, the effects of the different retinoids on three derived melanoma clones was dissimilar. For example, in clone A6, beta-all-transretinoic acid stimulated tyrosinase activity by 48% but caused only a 7% inhibition of cellular proliferation. This retinoid caused a more pronounced effect in the other two subclones, stimulating tyrosinase from 135 to 195% and inhibiting growth 19 to 33%. Al three melanoma clones demonstrated increased tyrosinase activity (110 to 225%) and reduced proliferation (37 to 52%) following exposure to 13-cis-retinoic acid. This retinoid was found overall to be the most effective stimulator of tyrosinase, while retinol was observed to be the least active, stimulating enzyme activity slightly (25%) in only one of three clones. Retinol inhibited proliferation 27 to 33% in two of three melanoma subclones. The aromatic retinoic acid ethyl ester elevated tyrosinase levels in two clones but inhibited the enzyme in one melanoma line. Cellular proliferation, however, was reduced in all three clones. These results suggest that retinoid-induced changes in human melanoma cell growth and differentiation reflect underlying cellular differences and diverse biochemical interactions.

Topics: Adolescent; Cell Differentiation; Cell Division; Cell Line; Clone Cells; Humans; Male; Melanins; Melanoma; Monophenol Monooxygenase; Tretinoin; Vitamin A

The presence and level of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) were determined in several neoplastic cell lines. These cells exhibited different degrees of susceptibility to growth inhibition in culture by two retinoids, retinyl acetate and retinoic acid. CRABP was detected in 10 and CRBP in 3 of the 11 tested cell lines. The levels of CRBP and CRABP were in the ranges 15-3,400 and 4-1,290 pmol per 10(9) cells, respectively, as determined by sucrose gradient centrifugation. Cell lines that contained CRABP included S91 and B16 melanomas; Mm5mT and DMBA No. 8 mammary adenocarcinomas; BW5147, BW5147.RicR, and P3 neoplastic lymphoid cells; F361.2 (a hybrid cell line obtained by fusion of MSV3T3 and BW5147); MSV3T3 sarcoma; and RAW8 lymphosarcoma. All but the last two cell lines were inhibited by retinoic acid in culture. CRBP was detected in extracts of S91, Mm5mT, and RAW8. Retinyl acetate inhibited the growth of all cell lines with the exception of RAW8, MSV3T3, and F361.2. No correlation was found between the level of either binding protein and the extent of growth inhibition by either retinyl acetate or retinoic acid. Neither of the binding proteins was detected in L1210-A5 leukemia cells, whose proliferation can be inhibited by both retinyl acetate and retinoic acid. These data indicated that screening cell lines for the presence and level of CRBP and CRABP is not sufficient to predict the susceptibility of cultured cells to growth inhibition by retinoids.

Topics: Adenocarcinoma; Animals; Carrier Proteins; Cell Count; Cell Line; Leukemia; Lymphoma, Non-Hodgkin; Melanoma; Mice; Neoplasms, Experimental; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Sarcoma; Tretinoin

S91 melanoma cells, which are sensitive to retinoic acid and contain a cellular retinoic acid-binding protein (RABP), were used in an investigation of the possible correlation between the capacities of various retinoids to inhibit cell proliferation and to bind to the RABP. Each of 27 different retinoids was evaluated for growth-inhibitory activity by exposing S91 melanoma cells to various retinoid concentrations between 1 nM and 10 microM. Subsequently, the ability of 17 of these retinoids to compete with [3H]retinoic acid for binding to RABP was determined. The results demonstrate that in addition to retinoic acid, many retinoids are capable of inhibiting the proliferation of S91 melanoma cells, although some are considerably less active. A positive correlation was found between the abilities of retinoids possessing a free carboxyl group at carbon 15 to inhibit cell proliferation and to bind to RABP. The structure-activity relationships established with the S91 cells are compared with previous reports on the biological activities of various retinoids in other systems.

Topics: Animals; Binding, Competitive; Cell Division; Cell Line; Melanoma; Mice; Neoplasms, Experimental; Retinol-Binding Proteins; Structure-Activity Relationship; Tretinoin; Vitamin A

Topics: Breast; Breast Neoplasms; Cell Division; Cell Line; Dose-Response Relationship, Drug; Humans; Melanoma; Neoplasms, Experimental; Time Factors; Tretinoin; Vitamin A

We studied the effects of retinoids on the in vitro survival of melanoma colony-forming cells in biopsies obtained from ten patients with metastatic melanoma. The results indicate that specific retinoids reduce the ability of fresh human melanoma cells to form colonies in soft agar. The retinoids studied had differential effects on the survival of clonogenic melanoma cells, and these effects vary from patient to patient. The data provide support for the clinical trial of selected retinoids in micrometastatic and advanced melanoma.

Topics: Cell Division; Clone Cells; Dose-Response Relationship, Drug; Etretinate; Humans; Melanoma; Tretinoin; Vitamin A

Topics: Animals; Cell Division; Clone Cells; Drug Resistance; Lung Neoplasms; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasms, Experimental; Tretinoin

The in vitro proliferation of murine melanoma cell lines S91 and B16 was inhibited by retinoic acid and retinyl acetate. The inhibitory effects were dependent on retinoid concentration and increased from 55 and 30% at 10(-9) M retinoic acid to 85 and 82% at 10(-5) M retinoic acid for S91 and B16 melanoma cells, respectively. S91 melanoma cells were more sensitive than B16 melanoma cells to inhibition by either retinoid, and both cell lines were more sensitive to retinoic acid than to retinyl acetate. When exposed to 10(-5) M retinoic acid, the two cell types grew at the same rate as did control cells for 48 hours, whereupon the growth rates of retinoid-treated cells decreased. After 6 days, the number of cells in control cultures increased 140 times (S91 melanoma cells) and 265 times (B16 melanoma cells), whereas retinoic acid-treated cells increased only 14 times (S91 melanoma cells) and 40 times (B16 melanoma cells). The degree of growth inhibition by retinoic acid was not dependent on initial cell density. Cortisone and hydrocortisone failed to prevent or reduce the inhibitory effect of retinoic acid; the release of lysosomal acid phosphatase was not increased and the intracellular level of 3',5'-cyclic AMP in cells grown for 5 days in the presence of 10(-5) M retinoic acid was not elevated. Viability of S91 and B16 cells after 8 days' exposure to 10(-5) M retinoic acid was similar to that in control cultures. The reduced growth rate of retinoic acid-treated cells reversed to the control rate 48-72 hours after removal of retinoic acid from the growth medium.

Topics: Animals; Cell Division; Cell Line; Cortisone; Cyclic AMP; Diterpenes; Dose-Response Relationship, Drug; Drug Interactions; Hydrocortisone; Kinetics; Melanoma; Mice; Neoplasms, Experimental; Retinyl Esters; Tretinoin; Vitamin A

Topics: Animals; Antineoplastic Agents; Cell Division; Cell Line; Cell Transformation, Neoplastic; Diterpenes; DNA, Neoplasm; Mammary Neoplasms, Experimental; Melanoma; Neoplasm Proteins; Neoplasms, Experimental; Retinol-Binding Proteins; Retinyl Esters; Sarcoma, Experimental; Tretinoin; Vitamin A