metallothionein and Melanoma

Distinguishing primary cancer from a metastasis is a fundamental task in surgical pathology that has crucial therapeutic and prognostic implications. When metastatic melanoma involves the skin and extends into the epidermis (epidermotropic), differentiating it from primary cutaneous melanoma may be particularly difficult. Early investigators dismissed this issue as a nonproblem and dogmatically declared that junctional change was requisite for the diagnosis of primary cutaneous melanoma. Epidermotropic metastases of malignant melanoma (EMMM) are now recognized to have many features in common with primary melanoma, which can include involvement of the dermal-epidermal junction, extension of the intraepidermal component beyond the dermal component, and even an epidermal-only pattern. This article traces the evolution in criteria used to diagnose EMMM and highlights why much of the original thinking is no longer accepted. Current concepts in EMMM morphology are elaborated and illustrated. The role of immunomicroscopy and molecular pathology in the detection of EMMM is also discussed.

Topics: Adult; Diagnosis, Differential; DNA, Neoplasm; Epidermis; Female; Humans; Intercellular Adhesion Molecule-1; Male; Melanoma; Metallothionein; Middle Aged; Neoplasms, Multiple Primary; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms

Zinc is a feature trace element of pigment cells and tissues. Organelles, in which melanin is synthesized and stored, i.e. melanosomes, represent a zinc reservoir at the subcellular level. In order to understand function of metals in tissues, cells and their constituents, knowledge is needed on metal interactions with intracellular targets. The possible zinc ligands in pigment cells include melanin, metallothionein, melanotransferrin, B700 and related proteins, ferritin, zinc enzymes and low molecular weight ligands. Areas of a special interest in relation of pigment cells and structures to zinc--such as zinc effect on melanogenesis, zinc excretion and buffering by melanosomes, zinc function in free radical processes as well as zinc role in melanomas--have been reviewed. High level of zinc in pigment cells may indicate a physiological defense against the potential danger of oxidative stress.

Topics: Animals; Humans; Ligands; Melanins; Melanocytes; Melanoma; Metallothionein; Pigmentation; Zinc

When a CpG island (CGI; a dense cluster of CpGs) located in the 5' region of a gene is methylated, its transcription is suppressed. Tumorigenesis of melanoma is associated with trace elements. Metallothionein 1A is closely associated with the metabolism of trace elements. However, little is known about the metallothionein 1A gene (MT1A) in melanoma.. The purpose is to reveal the methylation and expression status of MT1A in melanoma.. Quantitative real-time methylation-specific PCR (RT-MSP) and bisulfite sequencing were performed to examine MT1A methylation status. Quantitative real-time reverse transcription-PCR (RT-PCR) was performed to examine MT1A expression.. Some melanoma cell lines exhibited high methylation levels of the CGI located in the 5' region of MT1A (5' MT1A CGI) with suppression of MT1A. Other melanoma cell lines and normal cultured melanocytes exhibited low methylation levels of 5' MT1A CGI with expression of MT1A. Treatment with a demethylating agent resulted in transcriptional induction of MT1A in the melanoma cell lines SK-MEL-5 and G-361 with high methylation levels prior to treatment. The methylation levels of 5' MT1A CGI ranged widely from 0.0% to 91.4% in 21 clinical melanoma samples but showed a narrow, low range from 0.0% to 6.4% in 23 clinical melanocytic nevus samples. Data of bisulfite sequencing was generally compatible with those of RT-MSP. The methylation levels ranged according to the types of melanoma (Kruskal-Wallis test, P=0.047).. MT1A is aberrantly silenced by DNA methylation of 5' MT1A CGI in melanoma.

Topics: 5' Untranslated Regions; Adolescent; Adult; Aged; Aged, 80 and over; Azacitidine; Cell Line, Tumor; Child; CpG Islands; Decitabine; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Male; Melanocytes; Melanoma; Metallothionein; Middle Aged; Nevus, Pigmented; Real-Time Polymerase Chain Reaction; Skin Neoplasms; Young Adult

Reversing the dysfunctional T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we analyzed population and single-cell RNA profiles of CD8(+) tumor-infiltrating lymphocytes (TILs) and used genetic perturbations to identify a distinct gene module for T cell dysfunction that can be uncoupled from T cell activation. This distinct dysfunction module is downstream of intracellular metallothioneins that regulate zinc metabolism and can be identified at single-cell resolution. We further identify Gata-3, a zinc-finger transcription factor in the dysfunctional module, as a regulator of dysfunction, and we use CRISPR-Cas9 genome editing to show that it drives a dysfunctional phenotype in CD8(+) TILs. Our results open novel avenues for targeting dysfunctional T cell states while leaving activation programs intact.

Topics: Animals; Carcinogenesis; CD8-Positive T-Lymphocytes; CRISPR-Cas Systems; Female; GATA3 Transcription Factor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Melanoma; Metallothionein; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL

The present study evaluates the impact of immune cell populations on metastatic development in a model of spontaneous melanoma [mice expressing the human RET oncogene under the control of the metallothionein promoter (MT/ret mice)]. In this model, cancer cells disseminate early but remain dormant for several weeks. Then, MT/ret mice develop cutaneous metastases and, finally, distant metastases. A total of 35% of MT/ret mice develop a vitiligo, a skin depigmentation attributable to the lysis of normal melanocytes, associated with a delay in tumor progression. Here, we find that regulatory CD4(+) T cells accumulate in the skin, the spleen, and tumor-draining lymph nodes of MT/ret mice not developing vitiligo. Regulatory T-cell depletion and IL-10 neutralization led to increased occurrence of vitiligo that correlated with a decreased incidence of melanoma metastases. In contrast, inflammatory monocytes/dendritic cells accumulate in the skin of MT/ret mice with active vitiligo. Moreover, they inhibit tumor cell proliferation in vitro through a reactive oxygen species-dependent mechanism, and both their depletion and reactive oxygen species neutralization in vivo increased tumor cell dissemination. Altogether, our data suggest that regulatory CD4(+) T cells favor tumor progression, in part, by inhibiting recruitment and/or differentiation of inflammatory monocytes in the skin.

Topics: Animals; Cell Proliferation; Female; Flow Cytometry; Humans; Inflammation; Lymph Nodes; Male; Melanoma; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Monocytes; Neoplasm Metastasis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-ret; Reactive Oxygen Species; Skin; Spleen; T-Lymphocytes, Regulatory; Time Factors; Vitiligo

The formation of metastases and the efficacy of systemic therapies in cutaneous malignant melanoma (CMM) depend on the characteristics of the tumour cells and the host immune response. Aberrant expression of metallothionein (MT) has been observed in several types of cancers with poor prognoses.. To perform an immunohistochemical study on primary CMM comparing the MT expression of tumours without metastases (n = 23) to that of samples with haematogenous metastases (n = 23) and to examine the correlation between MT staining and immunological markers relevant in CMM progression.. The immunohistochemical labelling of different tumour sections was analysed using tissue microarrays for the evaluation of the suitability of this method in future studies.. Our results suggest that MT overexpression is significantly more frequent in primary CMM with haematogenous metastases (P = 0.018) and that the overexpression is independent of the Breslow tumour thickness (R = 0.102, P = 0.501). Interestingly, MT overexpression of the tumour cells was correlated with the presence of tumour-infiltrating CD68(+) macrophages (P = 0.003), a known predictive factor for melanoma progression, thereby suggesting a role for MT in the development of a defective host immune response. Furthermore, the presence of CD163(+) macrophages infiltrating the tumours correlated with metastasis formation (P < 0.001), whereas the presence CD1a(+) dendritic cells surrounding the tumours was associated with a lower risk of haematogenous spread (P = 0.003).. Our results demonstrate that MT may represent a suitable prognostic factor that can characterize the metastasising ability of CMM and the tumour-promoting host immune response.

Topics: Antigens, CD; Disease Progression; Female; Humans; Macrophages; Male; Melanoma; Metallothionein; Neoplasm Metastasis; Risk Factors; Skin Neoplasms

Multiple functional implications have been suggested for a limited number of chemokines and their cognate receptors in melanoma pathogenesis. The purpose of this study was to investigate the potential role of the chemokine receptors CXCR4, CCR7, CCR9, and CCR10 as prognostic markers in human primary cutaneous melanoma. Chemokine receptor expression was analyzed by immunohistochemistry in a total of 38 patients with cutaneous melanoma. Results were statistically correlated with melanoma features and clinical disease progression. No significant correlation between overexpression of CXCR4 or CCR9 and survival or prognosis was found. CCR7 overexpression was associated with significantly lower survival (0.005 log rank) and shorter time to progression (0.009 log rank)-similar to CCR10 overexpression (lower survival: 0.001 log rank, shorter time to progression: 0.002 log rank). In addition, CCR7 overexpression correlated with expression of metallothionein, while CCR10 seems to be associated with cerebral metastases. Two chemokine receptors permitting the identification of high-risk patients were identified: CCR7 and CCR10 overexpressions were found to be associated with a worse outcome of disease course independent of Breslow's tumor thickness and Clark level, thus representing possible additional prognostic markers.

Topics: Biomarkers; Disease Progression; Female; Humans; Male; Melanoma; Metallothionein; Prognosis; Receptors, CCR; Receptors, CCR10; Receptors, CCR7; Receptors, CXCR4; Severity of Illness Index; Skin Neoplasms

Tumors affect myelopoeisis and induce the expansion of myeloid cells with immunosuppressive activity. In the MT/ret model of spontaneous metastatic melanoma, myeloid cells are the most abundant tumor infiltrating hematopoietic population and their proportion is highest in the most aggressive cutaneous metastasis. Our data suggest that the tumor microenvironment favors polarization of myeloid cells into type 2 cells characterized by F4/80 expression, a weak capacity to secrete IL-12 and a high production of arginase. Myeloid cells from tumor and spleen of MT/ret mice inhibit T cell proliferation and IFNγ secretion. Interestingly, T cells play a role in type 2 polarization of myeloid cells. Indeed, intra-tumoral myeloid cells from MT/ret mice lacking T cells are not only less suppressive towards T cells than corresponding cells from wild-type MT/ret mice, but they also inhibit more efficiently melanoma cell proliferation. Thus, our data support the existence of a vicious circle, in which T cells may favor cancer development by establishing an environment that is likely to skew myeloid cell immunity toward a tumor promoting response that, in turn, suppresses immune effector cell functions.

Topics: Animals; Antigens, Differentiation; CD11b Antigen; Cell Proliferation; Disease Models, Animal; Disease Progression; Female; Flow Cytometry; Humans; Interferon-gamma; Interleukin-10; Interleukin-12; Male; Melanoma; Metallothionein; Mice; Mice, Knockout; Mice, Transgenic; Myeloid Cells; Proto-Oncogene Proteins c-ret; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Tumor Microenvironment

DNA methylation plays a major role in cancer by silencing tumour suppressor genes. In melanoma, only a discrete number of methylated genes have been identified so far. After the treatment of melanoma cells with a DNA methyltransferase inhibitor and subsequent transcriptomic profiling, we had identified earlier a cohort of melanoma progression-associated genes regulated by methylation. Here, we identified which of these genes are directly methylated in melanoma cell lines and tissues. First, we examined 16 genes by bisulphite sequencing in the WM793 isogenic cell line model series. Five of these genes (CYBA, FABP5, MT1E, TSPY1 and TAC1) displayed increased methylation in several invasive cell lines compared with the parental WM793 cells, indicating their involvement in progression. Next, we analyzed several matched primary/metastatic tumours using methylation-specific PCR, which revealed that MT1E (one of the five genes assessed) was methylated in the largest proportion of tumours. Examination of a larger cohort of samples showed that 1 of 17 (6%) of the benign naevi, 16 of 43 (37%) primary tumours and 6 of 13 (46%) of the metastases displayed MT1E methylation. In addition, ectopic over-expression of MT1E mediated sensitization to cisplatin-induced apoptosis. Overall, these studies suggest that MT1E is a potential tumour suppressor gene, whose loss may promote resistance to apoptosis-inducing therapies.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cells, Cultured; Cisplatin; DNA Methylation; Drug Resistance, Neoplasm; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Melanoma; Metallothionein; Microarray Analysis; Skin Neoplasms; Transfection

Malignant melanoma is one of the most aggressive human neoplasms and its incidence is still increasing. Prognoses for melanoma patients are currently based on statistical parameters. For estimating the risk for a possible progression and for overall survival, Breslow tumour thickness and the invasion level (Clark level) are the most established markers for melanomas at the time of the primary diagnosis. In thicker melanomas (>1 mm) the additional information about the status of the sentinel lymph-node (SLN) might be helpful. Nevertheless new prognostic parameters are needed, that will allow us to formulate more precise prognoses for the individual cases. The metallo-thionein family is a class of intracellular low-molecular-weight, cysteine-rich proteins with a high affinity for heavy-metal ions. They are involved in many (patho-) physiological processes and presumably play an important role in the carcinogenesis. In the last decades overexpression of immunohistochemically labelled metallothioneins (MTs) on paraffin-embedded tissues turned out as a highly significant prognostic marker in different tumours. This review summarizes the results of those studies, in which MT-overexpression was able to show a very high significance for progression and survival in melanoma patients. In contrast to most other progression markers, MT-overexpression is independent from tumour-thickness, and is highly specific even in thin (low risk) melanoma patients. Nowadays, in high risk melanoma patients sentinel lymph-node (SLN-) biopsy is performed, a surgical technique with predictive value for progression, the benefit of this procedure for the individual overall survival still remains unclear. In a study comparing SLN and MT-overexpression the results corroborate the validity of MT-overexpression in primary melanoma as a useful additional prognostic marker, accuracy is comparable although to some degree supplementary to the results of SLN biopsy.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Child; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Male; Melanoma; Metallothionein; Middle Aged; Neoplasm Staging; Predictive Value of Tests; Prognosis; Retrospective Studies; Sentinel Lymph Node Biopsy; Skin Neoplasms; Up-Regulation

DNA methylation is an important component of epigenetic modifications, which influences the transcriptional machinery aberrant in many human diseases. In this study we present the first genome-wide integrative analysis of promoter methylation and gene expression for the identification of methylation markers in melanoma. Genome-wide promoter methylation and gene expression of eight early-passage human melanoma cell strains were compared with newborn and adult melanocytes. We used linear mixed effect models (LME) in combination with a series of filters based on the localization of promoter methylation relative to the transcription start site, overall promoter CpG content, and differential gene expression to discover DNA methylation markers. This approach identified 76 markers, of which 68 were hyper- and eight hypomethylated (LME, P < 0.05). Promoter methylation and differential gene expression of five markers (COL1A2, NPM2, HSPB6, DDIT4L, MT1G) were validated by sequencing of bisulfite-modified DNA and real-time reverse transcriptase PCR, respectively. Importantly, the incidence of promoter methylation of the validated markers increased moderately in early and significantly in advanced-stage melanomas, using early-passage cell strains and snap-frozen tissues (n = 18 and n = 24, respectively) compared with normal melanocytes and nevi (n = 11 and n = 9, respectively). Our approach allows robust identification of methylation markers that can be applied to other studies involving genome-wide promoter methylation. In conclusion, this study represents the first unbiased systematic effort to determine methylation markers in melanoma and revealed several novel genes regulated by promoter methylation that were not described in cancer cells before.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Biomarkers, Tumor; Cells, Cultured; Cluster Analysis; Collagen; Collagen Type I; DNA Methylation; Female; Gene Expression Profiling; Genome-Wide Association Study; Genome, Human; HSP20 Heat-Shock Proteins; Humans; Infant, Newborn; Male; Melanoma; Metallothionein; Middle Aged; Molecular Chaperones; Nuclear Proteins; Nucleoplasmins; Oligonucleotide Array Sequence Analysis; Phosphoproteins; Promoter Regions, Genetic; Proteins; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Tumor Cells, Cultured

Metallothioneins (MT) are ubiquitous, intracellular small proteins with high affinity for heavy metal ions. Immunohistochemical MT overexpression in paraffin-embedded tissues of patients with primary melanoma is associated with poor prognosis. While sentinel lymph node (SLN) biopsy is an established surgical technique for high-risk melanoma patients with predictive value for progression, the benefit of this procedure for the individual patient's overall survival remains unclear.. We examined the role of MT overexpression in comparison with SLN biopsy in melanoma patients as a prognostic marker for progression and survival. One hundred and fifty-eight (158) patients underwent SLN biopsy due to high-risk melanoma. Primary melanoma specimens were investigated by using a monoclonal antibody against MT on routinely fixed, paraffin-embedded tissues. The patients were followed up (median 37 months); the data of disease free survival and overall survival were calculated with a broad panel of statistical analyses.. Twenty-eight (18%) out of 158 recruited melanoma patients developed metastases, 17 (11%) patients died due to widespread disease. Kaplan-Meier curves gave significant disadvantages for the MT-positive as well as the SLN-positive group for progression and survival. In the Fisher's exact test and Pearson's chi(2)-test MT overexpression was highly significant for progression, whereas SLN biopsy failed significance. In univariate as well as multivariate Cox regression analysis MT overexpression proved an excellent marker for progression (P=0.007 and P=0.009), although the P-values for survival were not significant. In contrast, while in the univariate analysis SLN biopsy did not show significant results for progression it did for survival, and in the multivariate analysis reached a P-value < 0.05 for both measured endpoints.. Results corroborate the validity of MT overexpression in primary melanoma as a useful prognostic marker in melanoma patients. Accuracy is comparable and to some degree supplementary to the results of SLN biopsy.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Chi-Square Distribution; Child; Disease Progression; Female; Humans; Immunohistochemistry; Male; Melanoma; Metallothionein; Middle Aged; Predictive Value of Tests; Prognosis; Proportional Hazards Models; Sentinel Lymph Node Biopsy; Skin Neoplasms; Survival Analysis

Topics: Disease-Free Survival; Humans; Immunohistochemistry; Melanoma; Metallothionein; Prognosis; Prospective Studies; Skin Neoplasms

Metallothioneins (MT) are ubiquitous, intracellular small proteins with high affinity for heavy metal ions. In the last decades, it was shown that MT overexpression in a variety of cancers is associated with resistance to anticancer drugs and is combined with a poor prognosis. In this prospective study, we examined the role of MT overexpression in melanoma patients as a prognostic factor for progression and survival. Between 1993 and 2004, 3386 patients with primary cutaneous melanoma were investigated by using a monoclonal antibody against MT on routinely fixed, paraffin-embedded tissues. In all, 1270 patients could be followed up for further statistical analysis (Fisher's exact test, Mantel-Haenszel chi2 test, Kaplan-Meier curves). The MT data of disease-free interval and overall survival were compared univariately and multivariately in Cox regression analysis. Immunohistochemical overexpression of MT in tumour cells of patients with primary melanoma (310 of 1270; 24.4%) was associated with a higher risk for progression (117 of 167; 70.1%) and reduced survival (80 of 110; 72.7%) of the disease (P<0.0001). Similarly, Kaplan-Meier curves gave highly significant disadvantages for the MT-positive group. Univariate analysis (relative risk 7.4; 95% confidence interval (CI) 5.2-10.2; P<0.0001 for progression; relative risk 7.1; 95% CI 4.7-10.9; P<0.0001 for survival), as well as multivariate analysis with other prognostic markers resulted in MT overexpression as a highly significant and independent factor for prognosis in primary melanoma.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Biomarkers, Tumor; Child; Cohort Studies; Female; Gene Expression Profiling; Humans; Immunohistochemistry; Male; Melanoma; Metallothionein; Middle Aged; Multivariate Analysis; Prognosis; Prospective Studies; Skin Neoplasms; Survival Analysis; Up-Regulation

Metallothioneins (MTs) are ubiquitous proteins with high affinity for heavy metal ions, e.g. zinc, copper and cadmium. In the last decade it has been shown that MT overexpression in a variety of cancers is associated with resistance to anticancer drugs and radiotherapy, and with a poor prognosis.. To examine the role of MT overexpression in melanoma patients as a prognostic factor for progression and survival.. In a prospective cohort study 760 patients with primary cutaneous melanoma were investigated over 5 years (1993-98) by using a monoclonal antibody (E9) against MT on routinely fixed and paraffin-embedded tissues. In total, 520 patients were able to be followed up for progression of their disease or death due to melanoma and were included for statistical analysis (Fisher's exact test, Mantel-Haenszel chi2 test, Kaplan-Meier curves). MT data, progress-free interval and overall survival were compared univariately and multivariately with other prognostic factors, e.g. Breslow thickness, Clark level, ulceration, localization, age and gender (Cox regression analysis).. The immunohistochemical overexpression of MT in tumour cells (cut-off level > 10% of all tumour cells) in patients with primary melanoma (156 of 520; 30%) was associated with a higher risk for progression of the disease (33 of 45; 73%) and reduced survival (24 of 30; 80%) than MT-negative lesions [364 of 520 (70%), 12 of 45 (27%) and six of 30 (20%), respectively (P < 0.0001)]. Similarly, Kaplan-Meier tumour-free survival and overall survival curves for the comparison of MT-positive and MT-negative tumours gave highly significant advantages for the MT-negative group. In a univariate analysis (comparison with Breslow thickness: relative risk 2.9, 95% confidence interval, CI 1.46-5.76, P = 0.0023 for progression; relative risk 4.19, 95% CI 1.73-10.19, P = 0.0015 for survival), as well as in a multivariate analysis with other prognostic markers, MT overexpression turned out to be a highly significant and independent factor for prognosis in primary melanoma.. MT overexpression in primary melanoma is associated with an increased risk for disease progression. This marker is independent from Breslow thickness and helps to identify those thin melanomas (< 1.5 mm) that are at increased risk of progression. Moreover, the immunohistochemical staining of paraffin material is a cheap, easy and widely available technique to gain these results.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Disease Progression; Female; Humans; Immunohistochemistry; Male; Melanoma; Metallothionein; Middle Aged; Prognosis; Proportional Hazards Models; Prospective Studies; Risk Factors; Skin Neoplasms; Survival Analysis; Survival Rate

Recently, Zelger et al. found that metallothionein expression in melanoma is a useful prognostic indicator in white patients. In this study, we evaluated metallothionein expression in patients with melanoma as a prognostic indicator. We studied the tumors of 44 patients with cutaneous melanoma seen in our clinic from July 1988 to August 1998. Twenty-five neoplasms were metallothionein-positive, and 19 were metallothionein-negative. Only 9 (37.5%) of 24 cases of level I through III melanoma were positive for metallothionein, but 16 (80%) of 20 level IV and V cases were positive. Eight (40%) of 20 cases of "thin" melanoma (thickness: < or = 1.5 mm) were metallothionein-positive, and 17 (70.8%) of 24 cases of "thick" melanoma (thickness: > 1.5 mm) were metallothionein-positive. These results indicate a strong correlation of metallothionein expression with the level of invasion and tumor thickness in melanoma. The survival distribution function curve (Kaplan-Meier) for metallothionein expression showed a much better survival rate in the metallothionein-negative group than in the metallothionein-positive group.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Child, Preschool; Female; Follow-Up Studies; Humans; Immunohistochemistry; Infant; Male; Melanoma; Metallothionein; Middle Aged; Nevus, Pigmented; Skin Neoplasms; Survival Analysis; Survival Rate

We recently established a metallothionein-I(MT)/RET transgenic mouse line in which skin melanosis, benign melanocytic tumor and malignant melanoma develop stepwise. Malignant melanoma cells but not benign melanocytic tumor cells had metastatic ability in transgenic mice. In the present study, we investigated the expression of several matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), including MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MT1-MMP, TIMP-1 and TIMP-2, in these tumors. Western and northern blot analyses revealed that malignant transformation of melanocytic tumors developed in MT/RET transgenic mice accompanied with upregulation of MMP-9 and downregulation of TIMP-2. Expression of other MMP and TIMP genes examined was very low or undetectable in both benign and malignant tumors. Since activation of MMP-9 in malignant tumors was detected by gelatin zymography, these results suggest that imbalance of expression of the MMP-9 and TIMP-2 genes might be associated with metastatic ability of melanoma cells developed in MT/RET transgenic mice.

Topics: Animals; Cell Transformation, Neoplastic; Collagenases; Drosophila Proteins; Gelatinases; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; In Situ Hybridization; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Melanocytes; Melanoma; Metalloendopeptidases; Metallothionein; Mice; Mice, Transgenic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ret; Proto-Oncogenes; Receptor Protein-Tyrosine Kinases; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms

Because metallothionein (MT) is elevated and may be protective in UV-irradiated skin, we have studied the effects of UV and other agents on MT transcription using the sheep MT 1A promoter, linked to the beta-galactosidase gene and stably transfected into human cell lines. beta-galactosidase reporter activity was inducible by adding Zn2+ ions to the medium (100 microM for 2-4 h). Two differentiating agents, butyric acid and azelaic bishydroxamic acid (ABHA), significantly increased the response to Zn2+ in a melanoma cell line (MM96L-gal). UVB (280-315 nm) had two distinct, time-dependent effects. During the first 4 h after irradiation, high doses of UVB inhibited induction by Zn2+, an effect that was made more acute by simultaneous exposure to the differentiating agents. These changes in reporter activity were not due to alterations in Zn2+ transport into the cell. The UVB-depressed MT response subsequently recovered and by 24 h was double the control, yet remained sensitive to ABHA. Reporter activity in transfected HeLa cells differed from that in MM96L, being depressed 4 and 24 h after UVB and insensitive to ABHA at both times. Galactosidase reporter activity driven by non-MT promoters was not affected by these treatments. Dependence of MT transcriptional activity on UV-related DNA damage could be inferred because equitoxic UVC (254 nm) affected the response to Zn2+ in a similar fashion, whereas UVA, cisplatin and a methylating agent had no effect. The MT response was partly dependent on the PKC signal transduction pathway because it was inhibited by phorbol ester in HeLa, and by bisindolyl maleimide in HeLa and MM96L. The biphasic MT transcriptional response may model a signal transduction pathway that gives an early, depressed response to acute UV damage, with exacerbation by concurrent differentiation stimuli, but switches to a positive, cell-specific and potentially protective response at later times.

Topics: Animals; Cell Differentiation; HeLa Cells; Humans; Melanoma; Metallothionein; Promoter Regions, Genetic; Sheep; Skin Neoplasms; Tumor Cells, Cultured; Ultraviolet Rays

Topics: Adult; Aged; Child; Child, Preschool; Diagnosis, Differential; Female; Humans; Immunohistochemistry; Infant; Male; Melanoma; Metallothionein; Middle Aged; Nevus, Epithelioid and Spindle Cell; Skin Neoplasms

Tissue inhibitor of metalloproteinases (TIMPs) are secreted proteins that regulate the activity of metalloproteinases, enzymes important in development, tissue remodeling, angiogenesis, and tumorigenesis. To assess the importance of three highly conserved amino acids, His7, Asp16, and His95, in determining the biological properties of mouse TIMP-1, they were mutated into Arg, Tyr, and Arg, respectively. Recombinant vectors constructed to express the wild-type and mutant TIMP-1 proteins under the control of the metallothionein promoter were transfected into mouse melanoma B16F10 cells, which produce very little TIMP-1. Individual clones were isolated and characterized by Southern, Northern, and Western blotting to verify the presence of the TIMP-1 minigene and its expression. Analyses of conditioned media for collagenase-inhibiting activity indicated that both histidine mutants, but not the aspartic acid mutant, were functionally impaired. An investigation of the cell migration, matrix invasion, and tumor formation capabilities of several individual clones representing each of the mutants revealed that the His7Arg and His95Arg mutations, but not the Asp16Tyr mutation, largely abolished the ability of the protein to inhibit all of these activities. These data establish that for B16F10 cells, endogenously generated TIMP-1 is an effective inhibitor not only of matrix invasion and tumorigenicity but also, unexpectedly, of cell motility on plastic. The novel finding that both His7 and His95 are separately essential for significant TIMP-1 activity in vivo provides an important new insight into TIMP-1 function.

Topics: Amino Acid Sequence; Animals; Cell Movement; Enzyme Inhibitors; Genetic Vectors; Glycoproteins; Glycosylation; Histidine; Male; Matrix Metalloproteinase Inhibitors; Melanoma; Metallothionein; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Mutagenesis, Site-Directed; Neoplasm Invasiveness; Promoter Regions, Genetic; RNA, Messenger; RNA, Neoplasm; Tissue Inhibitor of Metalloproteinases; Tumor Cells, Cultured

Topics: Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Immunoenzyme Techniques; Melanoma; Metallothionein; Skin Neoplasms

Metallothioneins are ubiquitous proteins with a high affinity for heavy metal ions, e.g. zinc, copper and cadmium. Experimentally, metallothionein over-expression in cell lines derived from a variety of cancers has been associated with resistance to anticancer drugs and irradiation therapy. Using a monoclonal antibody (E9) to metallothionein we investigated immunoreactive expression in routinely fixed and paraffin-embedded tissue from 63 cases of malignant melanoma and 13 secondary deposits. Whereas a variety of cells in normal skin showed metallothionein expression, all forms of benign naevi studied were uniformly negative. In contrast 13/30 'thin' (< or = 1.5 mm; 0.7 +/- 0.4), 25/29 'thick' malignant melanoma (> 1.5 mm; 5.5 +/- 3.9) and 12/13 metastases were positive. Six patients with thin and 19 with thick melanoma with metallothionein expression died during a mean observation period of 6.4 +/- 1.8 and 3.6 +/- 2.5 years, respectively, their survival distribution function analyses giving statistically significant results for both the vertical tumour thickness (P < 0.0001) and metallothionein expression (P < 0.0001). These immunohistochemical results, based on routinely processed paraffin-embedded tissue, suggest that metallothionein expression in malignant melanoma is significantly associated with progressive disease and might therefore be a useful prognostic indicator.

Topics: Adult; Aged; Aged, 80 and over; Female; Humans; Immunohistochemistry; Male; Melanoma; Metallothionein; Middle Aged; Neoplasm Proteins; Nevus; Skin Neoplasms; Survival Analysis

Eleven pairs of surgically resected lung cancers and corresponding non-neoplastic lung tissue were evaluated for metallothionein (MT) and metal content (cadmium and copper) by the heme/109Cd binding assay and atomic absorption spectroscopy, respectively. Tissue samples, obtained from patients ranging in age from 51 to 79, included six adenocarcinomas, two small cell carcinomas, one mixed cell carcinoma, one squamous cell carcinoma, and one carcinoma of non-primary origin (i.e., melanoma). Paired t-tests showed that metallothionein and copper concentrations in lung tumor tissue were significantly elevated when compared to non-malignant lung tissue. Cu was the major metal associated with the 10 kDa MT fraction in lung tumors whereas Cd was the primary metal bound to MT from non-neoplastic lung tissue. Since Cu-thionein is also known to be elevated in fetal lung tissue, the possibility exists that MT might represent an oncodevelopmental product which is useful as a biomarker for the early detection of lung carcinoma.

Topics: Adenocarcinoma; Aged; Biomarkers, Tumor; Cadmium; Cadmium Radioisotopes; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Chromatography, Agarose; Copper; Humans; Lung; Lung Neoplasms; Melanoma; Metallothionein; Middle Aged; Neoplasm Proteins; Protein Binding; Spectrophotometry, Atomic

The varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein is thought to be the homolog of herpes simplex virus type 1 (HSV-1) ICP0, based on gene location and limited amino acid homology. However, HSV-1 ICP0 trans activates HSV-1 genes, while VZV ORF61 protein trans represses the function of VZV trans activators on VZV promoters in transient expression assays. To investigate the functional relatedness of HSV-1 ICP0 and VZV ORF61 protein, we established Vero and MeWo cell lines which stably express VZV ORF61 under the control of a metallothionein promoter and performed complementation studies with an HSV-1 ICP0 deletion mutant (7134). Mutant 7134 is impaired for plaque formation and replication at a low multiplicity of infection in cell culture, but these defects were complemented by up to 200-fold in Vero cell lines expressing VZV ORF61. Likewise, the efficiency of plaque formation was improved by up to 100-fold in MeWo cell lines expressing VZV ORF61. A cell line expressing another VZV immediate-early gene product (ORF62) was unable to complement mutant 7134. HSV-1 mutants which are deleted for other HSV-1 immediate-early gene products (ICP4, ICP27) were unable to grow in VZV ORF61-expressing cell lines. These results indicate that, despite marked differences in their sequences and in effects on their cognate promoters in transient expression assays, VZV ORF61 protein is the functional homolog of HSV-1 ICP0.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Blotting, Southern; Carrier Proteins; Chloramphenicol O-Acetyltransferase; Chlorocebus aethiops; Genes, Viral; Genetic Complementation Test; Herpesvirus 3, Human; Humans; Immediate-Early Proteins; Melanoma; Metallothionein; Molecular Sequence Data; Oligodeoxyribonucleotides; Open Reading Frames; Plasmids; Promoter Regions, Genetic; Repressor Proteins; Sequence Homology, Amino Acid; Simplexvirus; Transcriptional Activation; Tumor Cells, Cultured; Ubiquitin-Protein Ligases; Vero Cells; Viral Plaque Assay; Viral Proteins; Virus Replication

Previous studies have shown that the combination of dacarbazine, carmustine, cisplatin (DDP), and tamoxifen (TAM) produced a 53% overall response rate in patients with disseminated melanoma. Deletion of TAM from the regimen resulted in a fall in the response rate to 10%, and reincorporation of TAM returned the response rate to 52%, suggesting an important role for TAM. Using the human melanoma cell line T-289, we examined the nature of the interaction between TAM and each member of this combination in clonogenic assays in soft agar. The combination of TAM with DDP was highly synergistic as demonstrated by median effect analysis, whereas TAM was antagonistic with carmustine and an activated form of dacarbazine. The mean combination index at 50% kill was 0.26 +/- 0.02 (mean +/- SD) for TAM and DDP. This marked synergism was observed at concentrations of TAM that are clinically achievable. TAM had no effect on the uptake of the DDP analogue [3H]dichloro(ethylenediamine)platinum(II). There was no effect on the formation or repair of DDP intrastrand DNA adducts. Similarly, there was no effect demonstrated on the intracellular concentrations of glutathione or metallothioneins. We conclude that the interaction between TAM and DDP is truly synergistic in this cell line and is accomplished through none of the four mechanisms commonly associated with DDP resistance.

Topics: Carboplatin; Cisplatin; Dacarbazine; DNA; Drug Resistance; Drug Synergism; Humans; Melanoma; Metallothionein; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Tumor Cells, Cultured

The phenomenon of an elevated copper concentration in melanoma tumors was examined. It was demonstrated that 50-60% of total tissue copper is associated with metallothionein. The amino acid composition, electronic absorption and fluorescence were identical to that of the many known vertebrate Cu-thioneins. The immunological identification of melanoma tissue metallothionein was successful. The elevated Cu-thionein concentration in melanoma tumor tissue is not yet understood. It appears to be a common concept that in most tumors transient changes of the copper status parallel the metallothionein levels.

Topics: Amino Acids; Animals; Chromatography, Gel; Copper; Enzyme-Linked Immunosorbent Assay; Horses; Humans; Immunoblotting; Liver; Melanoma; Mercaptoethanol; Metallothionein; Mice; Mice, Inbred BALB C; Rats; Spectrophotometry

The molecular mechanisms of action of gold complexes in the treatment of rheumatoid arthritis are partially known, as are the mechanisms of action and potential utility of gold complexes in the treatment of neoplastic disease. In this paper, data relative to the mechanism of cytotoxicity and structure activity relationships are presented. Concepts of future research are also discussed.

Topics: Anti-Inflammatory Agents; Antibody Formation; Antineoplastic Agents; Arthritis, Rheumatoid; Auranofin; Aurothioglucose; Cell Survival; Cells, Cultured; DNA, Superhelical; Gold; Humans; Melanoma; Metallothionein; Structure-Activity Relationship