metallothionein and Carcinoma--Squamous-Cell

Schneiderian papillomas are uncommon tumors which may develop within the nasal cavity and comprise three well-defined histological types: sinonasal inverted papilloma (SNIP), exophytic papilloma, and oncocytic papilloma. It is well known the rate of Schneiderian papilloma may also present a malignant degeneration and SNIP represents the most important subgroup in consideration of its frequency and malignant propensity. Although HPV infection is always considered the first event favoring the development of SNIP, however, it is not established as an eventual connection between viral actions and malignant transformation. In fact, different molecular mechanisms are suspected to play a crucial role in this process and, currently, many authors agree that only by improving our knowledge about these mechanisms it will be possible to achieve new and effective targeted therapies. So the aim of this study was firstly to systematically review the literature focusing on different biomarkers that could be implicated in the stages of SNIP malignant degeneration. Secondly, a systematic review with meta-analysis was performed to better define the incidence of sinonasal malignancies originating from Schneiderian papilloma (SNIP, exophytic papilloma, and oncocytic papilloma). Twenty-nine studies comprising a total of 3177 patients were statistically analyzed. Results showed a 9% (95% CI = 7-11) overall rate of malignant transformation from Schneiderian papilloma. In conclusion, this analysis confirmed that the potential malignancy of Schneiderian papilloma should not be underestimated. On the other hand, our review showed the paucity of studies investigating the molecular alterations which may be related with the malignant transformation of SNIP.

Topics: Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cyclooxygenase 2; ErbB Receptors; Genetic Predisposition to Disease; Humans; Metallothionein; Molecular Targeted Therapy; Nasal Mucosa; Nose Neoplasms; Papilloma, Inverted; Papillomavirus Infections; Paranasal Sinus Neoplasms; Risk Factors

This review summarizes and evaluates the literature regarding the biomarkers for predicting the response and/or prognosis of esophageal squamous cell carcinoma (ESCC) patients treated with neoadjuvant chemoradiation therapy (CRT). There are seven categories of molecules known to correlate with the response and/or prognosis: tumor suppressors (p53, p21), cell cycle regulators (Cyclin D1, CDC25B, 14-3-3sigma), DNA repair molecules (p53R2, ERCC1), drug resistance proteins [metallothionein (MT)], angiogenic factors (VEGF), molecules involved in cell proliferation/invasion/metastasis (Ki-67, COX-2) and hedgehog signaling molecules (Gli-1). Of the above molecules, the tumor suppressor p53 is expected to be a representative biomarker for predicting the response and prognosis. The cell cycle markers CDC25B and 14-3-3sigma have potential as response biomarkers independent of the p53 status. The DNA repair markers, p53R2 or ERCC1, angiogenic molecule (VEGF), and hedgehog signaling pathway factor Gli-1 also have potential to predict the response and prognosis of ESCC. However, there are still many unanswered questions with regard to predicting the clinical effects of neoadjuvant CRT.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Cycle Proteins; Chemoradiotherapy, Adjuvant; Cyclooxygenase 2; DNA-Binding Proteins; Endonucleases; Esophageal Neoplasms; Forecasting; Humans; Ki-67 Antigen; Meta-Analysis as Topic; Metallothionein; Neoadjuvant Therapy; Prognosis; Ribonucleotide Reductases; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor A

The objective was to discover whether the oxygen-regulated protein, metallothionein, is expressed in the hypoxic cells of squamous cell carcinomas. Twenty patients with squamous cell carcinoma of the uterine cervix or head and neck were infused with a solution of the hypoxia marker, pimonidazole hydrochloride, at a dose of 0.5 g/m2. The following day, biopsies were collected, formalin fixed, paraffin embedded, and sectioned at 4 microm. Sections from each biopsy were immunostained for pimonidazole binding, metallothioneins I and II, involucrin, and proliferating cell nuclear antigen. A total of 84 biopsies were analyzed. Sixty-four of 84 biopsy sections contained hypoxia. Of the hypoxia-containing sections, 43 of 64 or 67% showed no microregional overlap between hypoxia and metallothionein; 7 of 64 showed overlap; and 14 of 64 showed a combination of overlap and no overlap. On a tumor-by-tumor basis, 5 of 7 head and neck and 7 of 13 cervix tumors showed no overlap between metallothionein and hypoxia at the microregional level. Ranges for the percentage of the area of hypoxia in head and neck (<0.9 to 17%) and cervix (<0.1 to 14%) tumors were similar. In the hypoxia-containing sections, immunostaining for involucrin, a molecular marker for differentiation, overlapped with that for hypoxia in 82% of the cases. The majority of hypoxic cells in squamous cell carcinomas do not express metallothionein protein, although metallothionein is induced by hypoxia in human tumor cells in vitro. Hypoxic cells in human tumors tend to be in regions immunostaining for involucrin, and it seems possible that differentiation of hypoxic cells in squamous cell carcinomas might affect metallothionein I and II expression.

Topics: Biomarkers; Carcinoma, Squamous Cell; Cell Hypoxia; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Metallothionein; Neoplasm Staging; Nitroimidazoles; Proliferating Cell Nuclear Antigen; Protein Precursors; Uterine Cervical Neoplasms

The invasive behaviour of squamous cell carcinoma (SCC), a common malignant tumour of the mouth, is a process mediated by cell proliferation, extracellular matrix proteolysis and other factors. Studies have shown a potential relationship between growth factors, metallothionein 2A (MT2A) and matrix metalloproteinase (MMP) activation in malignant tumours. The aim of this study was to downregulate MT2A in cells (Cal27) derived from human squamous cell carcinoma.. Cal27 cells with reduced MT2A were subjected to proliferation, migration and invasion assays. Immunofluorescence and western blot confirmed MT2A depletion by siRNA. Growth curve assays assessed cell proliferation. Indirect immunofluorescence analysed the expression of MT2A, MMP-2, MMP-9, epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), tumour necrosis factor alpha (TNF-α) and Ki67. Zymography evaluated the effects of MT2A silencing on MMP-2 and -9 expression. Migration and invasion activities were evaluated using migration and invasion assays.. CAL27 cells displayed MT2A, MMP-2, MMP-9, EGF, TGF-α, TNF-α and Ki67. MT2A depletion decreased MMP-9, EGF, TGF-α and Ki67 protein levels, while increasing TNF-α.. MT2A downregulation reduced cell proliferation, migration and invasion activities. Therefore, MT2A has an important role in cell proliferation, migration and invasion in human oral SCC cells.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Epidermal Growth Factor; Humans; Ki-67 Antigen; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metallothionein; Transforming Growth Factor alpha; Tumor Necrosis Factor-alpha

Cervical cancer represents one of the most important female genital cancers. Cervical squamous cell carcinoma (CESC) accounts for about 90% of all cervical malignancies and the prognosis are unsatisfied. Here we aimed to investigate the clinical relevance of metallothionein-like 5 (MTL5), a novel metallothionein-like protein, in CESC. RT-qPCR and immunohistochemistry staining showed that MTL5 was upregulated in CESC tissues than nontumorous cervix tissues, which is consistent with the data from TCGA database. Kaplan-Meier survival analysis revealed that higher MTL5 can help predict worse prognosis. In addition, Cox hazard regression analysis verified an independent predictive role of MTL5 in CESC. To further investigate the involvement of MTL5 in CESC, we conducted knockdown experiments in two CESC cell lines. As a result, silencing MTL5g significantly inhibited proliferation of CESC cells. Finally, we validated that silencing MTL5 can suppress CESC tumor growth in vivo using the mice subcutaneous xenografts model. Taken together, higher MTL5 indicates worse survival of CESC after surgical resection. Targeting MTL5 represents a potential therapy of CESC by inhibiting tumor growth, which deserves further investigations.

Topics: Animals; Carcinoma, Squamous Cell; Cell Proliferation; Female; Humans; Metallothionein; Mice; Prognosis; Uterine Cervical Neoplasms

Metallothioneins (MTs) are phylogenetically old cysteine-rich proteins, which are implicated in a variety of physiological and pathological processes. Their growth-regulating, anti-apoptotic and anti-inflammatory functions have been attributed not only to intracellular free radical scavenging and to zinc and copper regulation but also to the ability of secreted MT to bind on surface lipoprotein receptor-megalin/LRP2, which enables the endocytosis of MT-I/II and a wide range of other functionally distinct ligands. In the present study, we analysed the expression pattern of both proteins in 55 cases of premalignant transformation of cervical squamous cells, i.e. in low- and high-grade squamous intraepithelial lesion (LSIL and HSIL). The data showed that in LSIL (cervical intraepithelial neoplasia CIN1; N = 25) MTs were present only in basal and parabasal cells and that megalin was only weakly expressed. In HSIL (CIN2; N = 15 and CIN 3/carcinoma in situ; N = 15), however, overexpression and co-localization of MT with megalin were found in the entire hyperplastic epithelium. Moreover, megalin immunoreactivity appeared on the glandular epithelium and vascular endothelium, as well as on lymphatic cells in stroma. Besides, multiple megalin-positive cells expressed phosphorylated Akt1, implying that MT- and/or megalin-dependent prosurvival signal transduction pathways might contribute to the development of severe cervical dysplasia. The data emphasize the diagnostic power of combined MT/megalin analysis in pre-cancer screening.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Early Detection of Cancer; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Low Density Lipoprotein Receptor-Related Protein-2; Metallothionein; Transcriptome; Tumor Microenvironment; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

Metallothioneins (MTs) gene polymorphisms have been associated with the ability of free radical scavenging and detoxification of heavy metals leading to cancer development. Our aim was to revisit, in a Brazilian population, single-nucleotide polymorphisms (SNPs) of the MT gene family previously associated with oral squamous cell carcinoma (OSCC).. A case-control investigation with 28 OSCC patients and 45 controls was conducted, using conventional risk factors (tobacco use and alcohol consumption) as covariates. SNPs genotyping for rs8052334 (MT1B), rs964372 (MT1B), and rs1610216 (MT2A) was performed by PCR-RFLP, and SNPs for rs11076161 (MT1A) were analyzed by TaqMan assay.. The only SNP associated with increased risk for OSCC was the MT-1A AA genotype (OR = 4.7; p = 0.01). We have also evidenced for the first time a significant linkage disequilibrium between the SNPs of MT-2A and MT-1A in this population with the highest frequency (30%) of the unfavorable haplotype G/A/C/T (rs1610216 / rs11076161 / rs964372 / rs8052334) of MT gene polymorphisms (OR = 6.2; p = 0.04). Interestingly, after removing the effects of conventional risk factors, we have uncovered the significance of the AA genotype of the rs11076161 with increased odds of 19-fold higher towards OSCC development.. This is the first demonstration that a significant linkage disequilibrium among gene polymorphisms of the MT family may affect susceptibility to oral cancer, which is conditioned by the G/A/C/T haplotype (rs1610216/rs11076161/rs964372/ rs8052334) and the MT-1A gene polymorphism has a potential clinical utility for the OSCC risk assessment.

Topics: Brazil; Carcinoma, Squamous Cell; Case-Control Studies; Genetic Predisposition to Disease; Genotype; Head and Neck Neoplasms; Humans; Metallothionein; Mouth Neoplasms; Polymorphism, Single Nucleotide; Risk Factors; Squamous Cell Carcinoma of Head and Neck

The aim of this study was to identify the immunoexpression of methallothionein in oral squamous cell carcinoma as well as to address the correlation with clinical features, histological grade and patient survival. Samples were collected from 93 patients with tongue squamous cell carcinoma who presented for follow-up. Immunohistochemical expression of methallothionein in all groups was performed. The scoring system has previously been published by Tsurutani in 2005, which is based on intensity and distribution of staining. We used Kappa index to evaluate the degree of observers' agreement under metallothionein immunostaining and histological grade. Associations between methallothionein expression and clinical parameters (age, gender, smoking, tumor size, lymph node metastasis and disease stage) were examined for statistical significance using the chi-squared test. The overall survival rates were estimated by the Kaplan-Meier method and the relationship between protein expression and survival was compared using the log-rank test (p < 0.05). Our results showed no statistically significant association between methallothionein immunostaining and the selected clinicopathological variables. Immunohistochemistry results showed positive nuclear immunostaining for metallothionein in 62,37% (58/93) and negative for metallothionein 37,63% (35/93). The degree of examiners agreement by Kappa varied from substantial to perfect and both metallothionein immunostaining and histological grade were explored. The present study suggests that positive methallothionein expression found in tongue squamous cell carcinoma may not help to predict survival in the analyzed samples, as well as no relation between the protein and histological grade and clinical features was observed. In conclusion, the present study suggests that metallothionein is not associated with tongue squamous cell carcinoma clinicopathological characteristics and aggressiveness.

Topics: Adult; Aged; Aged, 80 and over; Biopsy; Carcinoma, Squamous Cell; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Metallothionein; Middle Aged; Neoplasm Grading; Neoplasm Staging; Paraffin Embedding; Time Factors; Tongue Neoplasms; Tumor Burden

Thioredoxin (Trx) and metallothionein (MT) are involved in the development of some carcinomas; however, the role of these proteins in labial carcinogenesis has not yet been tested. The aims of the study were to evaluate and to correlate the immunoexpression of Trx and MT in actinic cheilitis, lip squamous cell carcinoma, and normal vermillion lip mucosa.. Immunohistochemistry was undertaken for Trx and MT in samples of actinic cheilitis, lip squamous cell carcinoma, and normal lip mucosa. Qualitative and semi-quantitative evaluations were conducted. The proportion of stained cells, intensity of staining, and the cell compartment labeled were evaluated. A quickscore index was also calculated by multiplying the values of extension and intensity of nuclear and cytoplasmic staining, respectively, giving a maximum value of 9. Statistics were performed.. A remarkable nuclear Trx staining was seen in normal lip mucosa and cheilitis, not in carcinoma (p<0.05). Cytoplasmic Trx expression was widely detected in all lesions (p>0.05). MT was broadly expressed in nuclei and cytoplasm of carcinoma, but not in normal lip mucosa and cheilitis (p<0.05). Quickscores were in accordance with the qualitative results.. The current study showed a different immunopattern of Trx and MT between normal lip mucosa, actinic cheilitis and lip squamous cell carcinoma. The cellular compartment-based analyses evidenced differences that can be related to the proteins function. Considering the relevant roles of these proteins in cellular homeostasis, they seem to have an important role in lip carcinogenesis.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cheilitis; Female; Humans; Immunohistochemistry; Lip Neoplasms; Male; Metallothionein; Middle Aged; Neoplasm Grading; Thioredoxins

MCM2, MCM3 and MCM7 are minichromosome maintenance proteins found in dividing cells and they play a role in DNA synthesis. Increased MCM expression level is observed in cells of different cancer types. Additionally, metallothioneins (MT-I/II) are involved in control of cell proliferation and differentiation and changes of their expression are observed in many types of cancer. Ki-67 is known cancer cell proliferation antigen currently used in prognostic evaluation. The study material consisted of 83 laryngeal squamous cell cancer (LSCC) cases and 10 benign hypertrophic lesions of larynx epithelium as a control group. For the present study, laryngeal cancer cell line HEp-2 and human keratinocytes were employed, and to evaluate expression of all the markers, immunohistochemical method (IHC), immunofluorescence (IF) and western blot analysis were used. Statistical analysis showed strong positive correlation between expression of MCM2, MCM3, MCM7 and Ki-67 antigen in LSCC. Additionally, moderate positive correlation was observed between MCM3 and MT-I/II expression. In cancer cells, the level of expression of MCM3, MCM2, MCM7 and Ki-67 markers was increasing with the grade of LSCC malignancy. IF and western blot analysis showed higher MCM2, MCM3, MCM7 expression in HEp-2 cells in comparison to their expression in keratinocytes. MCM proteins might be useful markers of cell proliferation in LSCC.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Female; Head and Neck Neoplasms; Humans; Keratinocytes; Ki-67 Antigen; Laryngeal Neoplasms; Male; Metallothionein; Middle Aged; Minichromosome Maintenance Proteins; Squamous Cell Carcinoma of Head and Neck

Metallothioneins (MTs) are proteins associated with the carcinogenesis and prognosis of various tumours. Previous studies have shown their potential as biomarkers in oral squamous cell carcinoma (OSCC). Aiming to understand more clearly the function of MTs in OSCC we evaluated, for the first time, the gene expression profile of MTs in this neoplasm.. Tissue samples from 35 cases of tongue and/or floor of mouth OSCC, paired with their corresponding non-neoplastic oral mucosa (NNOM), were retrieved (2007-09). All tissues were analysed for the following genes using TaqMan(®) reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays: MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1X, MT2A, MT3 and MT4. The expression of MT1B and MT1H was seldom detected in both OSCC and NNOM. A significant loss of MT1A, MT1X, MT3 and MT4 expression and gain of MT1F expression was observed in OSCC, compared to NNOM. Cases with MT1G down-regulation exhibited the worst prognoses. The up-regulation of MT1X was restricted to non-metastatic cases, whereas up-regulation of MT3 was related to cases with lymph node metastasis.. Metallothionein mRNA expression is altered significantly in oral squamous cell carcinomas. The expression of MT1G, MT1X and MT3 may aid in the prognostic discrimination of OSCC cases.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Down-Regulation; Female; Humans; Male; Matrix Metalloproteinase 16; Metallothionein; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Prognosis; RNA, Messenger; RNA, Neoplasm; Tongue Neoplasms; Up-Regulation

The tumor microenvironment is made up of tissue that is responsible for the growth and progression of the tumor as well as its ability to initiate metastases. The cancer cells on the front of the tumor together with the macrophages and fibroblasts help to constitute the aggressive phenotype of the tumor. The presence of this aggressive phenotype is indicated by the local infiltration of cancer cells and by the development of lymph node metastases. In cases of uterine cancer, the extent of the local and distant spread of the disease is crucial for determining the type of therapeutic strategy to be applied - surgery alone, surgery followed by radio-chemotherapy, or radio-chemotherapy alone. In the interest of trying to improve the patient's quality of life, different studies supporting the therapeutic model of surgery alone have been conducted. While the cancer cells on the tumor front together with the macrophages and the fibroblasts help to constitute the aggressive phenotype of the tumor, metallothionein (MT) has been shown to have both pro-proliferative and anti-apoptotic activities and to participate in microenvironment remodeling. The aim of the current study was to determine the levels of MT immunoreactivity in the uterine cervical cancer cells as well as in the stromal fibroblasts and macrophages of the tumor microenvironment with respect to the depth of the local invasion and the extent of the distant metastases, so that its potential predictive value as a therapeutic strategy for cervical cancer can be ascertained.. We determined the levels of immunoreactivity of MT in a total of 57 carcinoma tissue samples (in the tumor front, in its central part, and in the macrophages and fibroblasts present within the tumor microenvironment). The patients from whom the samples derived were divided into groups with respect to the presence of lymph node metastases (distant spread) and to the depth of invasion (local spread) in relation to the FIGO stage.. Metallothionein immunoreactivity was observed in uterine cervical cancer cells; it was also identified in the fibroblasts and macrophages found within the microenvironments of the tumors of patients suffering from the disease. The MT immunoreactivity level significantly increased within the whole cancer nest in relation to the FIGO stage (intensity of the local spread of the disease). Similarly, the infiltration of MT-positive CAFs and TAMs statistically significantly increased in relation to the FIGO stage.. The level of MT immunoreactivity found in the fibroblasts and macrophages within the tumor microenvironment seems to be indicative of the intensity of the remodeled cervical tumor microenvironment, and this in turn may be related to the local advancement of the disease. Moreover, it appears that the intensity of the metallothionein immunoreactivity in the immunoreactivity profile of the cervical tumor may be linked to both the depth of the local invasion and the extent of the distant advancement of the disease.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Female; Fibroblasts; Humans; Lymphatic Metastasis; Macrophages; Metallothionein; Middle Aged; Tumor Microenvironment; Uterine Cervical Neoplasms

Metallothionein (MT) has been shown to have pro-proliferative anti-apoptotic activity and to be involved in microenvironment remodeling. The aim of this study has been to determine whether the changes in MT and vimentin immunoreactivity observed in cancer and its microenvironment are related to the local spread of the disease. The immunoreactivity levels of both MT and vimentin were evaluated together with CD56 and CD57 antigens in 49 tissue samples taken from patients with squamous cell carcinoma originating from the palatine tonsils and in 20 tissue samples derived from patients with chronic tonsillitis (the reference group). MT immunoreactivity levels were statistically significantly higher in the tissue samples from squamous cell carcinoma than in those of the reference group and also higher in the squamous cell carcinoma samples compared with the stromal samples. Moreover, stromal fibroblasts exhibited high vimentin and MT immunoreactivity levels. Statistically significantly higher MT immunoreactivity levels within the tumor cells were identified in patients with the presence of lymph node metastases in contrast to those patients without such metastases. Vimentin was detected in both the tumor and the stromal tissue samples and presented an interesting pattern of staining strongly expressed within the stroma and the septal architecture of the tumor. The number of CD56- and CD57-positive lymphocytes identified in tissue samples both from squamous cell carcinoma and from the stroma was statistically significantly lower than that in the reference group. MT expression by tumor cells is thus associated with an aggressive phenotype of the tumor and the ability to create metastases.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Female; Fibroblasts; Head and Neck Neoplasms; Humans; Male; Metallothionein; Middle Aged; Pharyngeal Neoplasms; Squamous Cell Carcinoma of Head and Neck; Vimentin

Metallothioneins (MT) are low-molecular weight proteins implicated in heavy metal detoxification, zinc and cooper homeostasis and cell protection against free radicals. In variety of cancers MT-overexpression was shown, but there are just a few studies on the role of MT in skin carcinogenesis. Current study was undertaken to evaluate MT and Ki-67 expression in pre-cancerous skin lesions as well as in fully developed skin cancers. 73 squamous cell carcinomas (SCC), 23 actinic keratoses (AK) and 20 normal skin samples were included in the study. In obtained paraffin sections immunohistochemical reactions were performed. MT-expression in SCC (mean 2.89 ± 1.83) was significantly higher than in AK (mean 1.69 ± 1.26)(p = 0.006) and higher than in normal skin (mean 2 ± 0.79) (p = 0.0075). The MT-expression positively correlated with Ki-67 expression (R = 0.28; p = 0.017) in SCC and in AK (R = 0.49; p = 0.018). Various clinico-pathological variables, e.g. morphology, size of lesions and the depth of neoplastic infiltration were not associated to MT-expression in both SCC and AK. The grade of histological differentiation of SCC correlated positively with Ki-67 antigen (p < 0.001) and did not correlate with MT-expression (p = 0.06). Ki-67 expression was higher in SCC and in AK than in healthy skin (p = 0,003). In SCC and in AK expression of Ki-67 antigen correlated positively with MT-expression (respectively p = 0.017 and p = 0.018). MT may serve as a good markers of proliferation in SCC and AK. MT-overexpression in SCC may suggest a potential role of MT in skin carcinogenesis.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Female; Humans; Immunohistochemistry; Keratosis, Actinic; Ki-67 Antigen; Male; Metallothionein; Middle Aged; Skin Neoplasms

To examine the presence of markers associated with malignancy, including p53, p21 cyclin-dependent kinase inhibitor 1A, murine double minutes-2, and others, in chronic hyperplastic candidiasis.. Immunohistochemical methods were used to examine the expression of p53, murine double minutes-2, p21 cyclin-dependent kinase inhibitor 1A, metallothionein, and proliferating cell nuclear antigen in 42 chronic hyperplastic candidiasis lesions and 11 non-infected control tissues. Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling was used to examine apoptosis, which was correlated with p53 expression. These markers were measured in lesions of chronic hyperplastic candidiasis that did not show any epithelial dysplasia or histological signs of malignancy.. p53 scores were higher in chronic hyperplastic candidiasis than in controls (P = 0.0046). Murine double-minutes 2 levels were not elevated. p21 cyclin-dependent kinase inhibitor 1A was increased in parabasal (P < 0.0001) and basal epithelial cells. Chronic hyperplastic candidiasis lesions showed a similar basal/parabasal metallothionein staining pattern to that seen in normal squamous epithelium. Proliferating cell nuclear antigen was increased (P = 0.0007), as was apoptosis (P = 0.0033).. Increased p53 in oral chronic hyperplastic candidiasis suggests an increased potential for malignant change in the epithelium, above that of normal tissues. Further functional investigation is required, as well as clinical follow-up studies.

Topics: Biomarkers, Tumor; Candidiasis, Oral; Carcinoma, Squamous Cell; Cyclin-Dependent Kinase Inhibitor p21; Epithelial Cells; Female; Gene Expression; Humans; Hyperplasia; Immunohistochemistry; In Situ Nick-End Labeling; Male; Metallothionein; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

We recently reported that TCRP1, a novel multidrug-resistance associated human gene, can mediate cisplatin resistance in OSCC cells. However, the molecular mechanism underlying this role of TCRP1 remained to be elucidated. In this study, by using Human Toxicology and Drug Resistance Microarray, we identified 30 genes with significantly different expression levels between Tca/PYM and TCRP1 knockdown cell lines. Co-immunoprecipitation experiments and GST-pull down assays showed that metallothionein1X (MT1X) and Akt interact with TCRP1. siRNA-mediated knockdown of TCRP1 and MT1X was found to sensitize cells to cisplatin, leading to increased apoptosis and inhibition of cell proliferation. These functions of TCRP1 may be caused at least in part via activation of the PI3K/Akt/NF-κB signaling pathway. Taken together, our findings indicate that TCRP1 may be an important drug target for improvement of the treatment and survival of patients with oral squamous cell carcinoma.

Topics: Antineoplastic Agents; Apoptosis; Base Sequence; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cisplatin; DNA Primers; Drug Resistance, Neoplasm; Gene Knockdown Techniques; Humans; Metallothionein; Mouth Neoplasms; NF-kappa B; Oligonucleotide Array Sequence Analysis; Proteins; Real-Time Polymerase Chain Reaction

We conducted an independent analysis of metallothionein 1 (MT-1) rs8052394, rs11076161, rs8052334, rs964372, rs7191779, and rs708274 in 587 individuals who were either healthy controls or subjects with oral squamous cell carcinoma (OSCC).. All participants provided a nucleic acid sample (blood) as well as epidemiologic information on covariates or "risk factors" for OSCC, including tobacco, alcohol, and areca quid use. The genotyping result was used in a logistic regression model that examined main effects as well as statistical interactions while controlling for confounders.. MT-1 is involved in regulation of zinc and copper homeostasis. It also is a potent antioxidant and its polymorphisms correlate with the risk for OSCC. Rs11076161 A, rs964372 C, and rs7191779 C alleles were protective against OSCC (adjusted OR = 0.53, 0.49, 0.36, respectively; p < 0.05), whereas rs8052394 A alleles were associated with increased risk. Areca quid chewing and tobacco use were strong risk factors for developing the disease and were associated with 20- and 8-fold increases in adjusted risk (p < 0.05), respectively.. Controlling for the effects of age, gender, areca quid, tobacco, and alcohol use, individuals with inherited the MT-1 rs11076161 AA, rs964372 CC, and rs7191779 GC genotypes may experience significant protection against OSCC, whereas individuals carrying the MT-1 rs8052394 A allele seem exposed to higher risk.

Topics: Alcohol Drinking; Areca; Carcinoma, Squamous Cell; Case-Control Studies; Female; Genotype; Humans; Male; Metallothionein; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Polymerase Chain Reaction; Polymorphism, Genetic; Prognosis; Risk Factors

Metallothionein (MT) has been implicated in several aspects of cancer pathobiology, such as differentiation, proliferation, apoptosis and invasion. The aim of the present study was to evaluate the clinical significance of MT expression in mobile tongue squamous cell carcinoma (SCC).. MT protein expression was assessed immunohistochemically on 49 mobile tongue SCC specimens, and was analysed in relation to clinicopathological characteristics, and overall and disease-free patient survival. All of the examined mobile tongue SCC cases showed MT positivity in tumour cells; however, neither MT overexpression nor staining intensity was significantly associated with clinicopathological parameters. MT cellular distribution was significantly associated with histopathological grade of differentiation and depth of invasion (P = 0.0188 and P = 0.0484, respectively). MT staining intensity was identified as a significant predictor of overall patient survival at both univariate (P = 0.0377) and multivariate (P = 0.0472) levels. Twenty-seven (55.10%) of the examined SCC cases showed MT positivity in squamous tongue epithelium adjacent to the tumour, the MT positivity being correlated with depth of invasion (P = 0.0281), vascular invasion (P = 0.0194), and the existence of lymph node metastases (P = 0.0194).. MT may be implicated in the development and progression of mobile tongue SCC and could be considered as a useful clinical marker for patient management and prognosis.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Disease-Free Survival; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Metallothionein; Middle Aged; Neoplasm Grading; Prognosis; Proportional Hazards Models; Tongue Neoplasms

This study aimed at finding out whether the expression of metallothionein (MT), laminin, Ki-67 antigen and minichromosome maintenance-2 (Mcm-2) protein changes with growing invasiveness of the tumour. The expression of these markers in primary tumours with no metastases to lymph nodes (PT N-) was compared with the expression in primary tumours with metastases in draining lymph nodes (PT N+). The difference in marker expression was also evaluated between metastatic lymph nodes (LN+) and the corresponding primary tumours (PT N+).. The studies were performed on tumour samples from 39 patients with squamous cell carcinoma of the oral cavity floor or of the oral part of the tongue. All the patients had been subjected to radical surgery, accompanied by the removal of lymph nodes. In 20 patients post-operative histopathology disclosed the presence of metastases in the draining lymph nodes (pN+), while in 19 patients the presence of such metastases was excluded (pN0).. The PT N+ group was found to contain a significantly higher percentage of cells with cytoplasmic expression of MT, than the PT N- group. In turn, a significant increase in the intensity of reaction of cytoplasmic MT and an increased percentage of cancer cells demonstrating MT expression in the cell nuclei was demonstrated in the LN+ compared to the PT N+ group. The expression of the remaining parameters did not significantly differ between PT N-, PT N+ and LN+.. A gradual increase in MT expression (both cytoplasmic and nuclear) takes place with progression of the tumour and the increased nuclear expression of MT in LN+ cells may suggest a role of MT in metastasis development in the studied tumours.

Topics: Adult; Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Cycle Proteins; Disease Progression; Female; Humans; Ki-67 Antigen; Lymphatic Metastasis; Male; Metallothionein; Middle Aged; Minichromosome Maintenance Complex Component 2; Mouth Neoplasms; Neoplasm Invasiveness; Nuclear Proteins

Metallothionein and p53 proteins have been associated with tumoral evolution and resistance against therapy. Experimentally, the former may modulate the activity of tumor suppressor protein through zinc exchange. However, there is no information on the relationship of these proteins in oral cancer.. Immunohistochemical detection of metallothionein and p53 antigens was performed in 100 oral squamous cell carcinomas. Results were compared to evaluate possible relationships between them and the disease-specific survival.. Mean cellular indexes of positivity were 66.5% and 52.5% for metallothionein and p53, respectively, and a positive correlation was found between them. Frequent nuclear metallothionein immunolocalization was associated to increased p53 expression. Concomitant overexpression of both antigens predicted shorter survival for patients with advanced disease.. These results corroborate the speculated association between metallothionein and p53 and suggest that simultaneous assessment of these proteins may be useful to evaluate aggressiveness of oral cancer.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Female; Humans; Immunoenzyme Techniques; Male; Metallothionein; Middle Aged; Mouth Neoplasms; Prognosis; Survival Rate; Tumor Suppressor Protein p53

Oral leukoplakia (OL) is the main potentially malignant lesion of the oral cavity, and oral squamous cell carcinoma (OSCC) accounts for more than 95% of all malignant neoplasms in the oral cavity. Therefore, the aim of this study was to verify the immunoexpression of p-Akt and Metallothionein (MT) proteins in dysplasic and neoplasic oral lesions.. Immunohistochemical studies were carried out on 10 normal epithelium, 30 OL and 15 OSCC paraffin-embedded samples. Immunoperoxidase reaction for p-Akt and MT proteins was applied on the specimens, and the positivity of the reactions was calculated for 1000 epithelial cells.. Using the ANOVA and the Tukey's post hoc statistical analyses, it was observed a significant difference in the immunoexpression for p-Akt and MT when the OSCC samples were compared with normal and dysplasic epithelial groups. In addition, the Pearson's correlation test showed a significant correlation between the proteins' expression.. Based on the data obtained, p-Akt and MT activation may play an important role in the conversion of a potentially malignant oral lesion to a malignant carcinoma since its earlier stages.

Topics: Adult; Analysis of Variance; Carcinoma, Squamous Cell; Case-Control Studies; Female; Humans; Leukoplakia, Oral; Male; Metallothionein; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Precancerous Conditions; Proto-Oncogene Proteins c-akt; Reference Values; Second Messenger Systems; Signal Transduction; Statistics, Nonparametric

The purpose of this study was to examine the effect of tricyclic antidepressant desipramine (DMI) on the growth inhibition and translocation of the glucocorticoid receptor (GR) from the cytoplasm to the nucleus in cancerous and noncancerous cell lines and the effect of DMI on GR-mediated transcription. Nontumorigenic, immortalized keratinocytes cell line (3PC), papilloma (MT1/2), and squamous cell carcinoma (Ca3/7) cell lines were initially used to study the cell growth inhibition by DMI. Although, the growth of all three cell lines was suppressed by DMI, it was more effective in Ca3/7 cells. Therefore, we next examined the effect of DMI on Ca3/7 cells, resistant to growth inhibition by the synthetic glucocorticoid fluocinolone acetonide (FA). DMI inhibited cell proliferation in a time-dependent manner. The translocation of GR was induced by FA alone, DMI alone, and combination of both agents. FA induced GR-mediated transcription in Ca3/7 cells transfected with a luciferase reporter gene under the control of glucocorticoid response element (GRE), but DMI alone did not affect GR-mediated transcription. However, DMI inhibited FA-induced, GR-mediated transcription when both agents were given together. Pretreatment with DMI followed by combination of DMI and FA decreased GR-mediated transcription more than pretreatment with FA. The expression of metallothionein-1 (Mt-1) gene, which is regulated by GR, was induced significantly by the combination of DMI and FA, and enhanced significantly by pretreatment with FA but not DMI. DMI is suggested to inhibit the growth of Ca3/7 cells and to affect GR-mediated transcription.

Topics: Animals; Anti-Inflammatory Agents; Antidepressive Agents, Tricyclic; Blotting, Western; Carcinoma, Squamous Cell; Cell Nucleus; Cell Transformation, Neoplastic; Cytoplasm; Desipramine; Fluocinolone Acetonide; Keratinocytes; Luciferases; Metallothionein; Mice; Papilloma; Receptors, Glucocorticoid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin Neoplasms; Transcription, Genetic

The incidence of esophageal squamous cell carcinoma (ESCC) is very high in northern China. This cancer has a very poor prognosis, mostly because it is usually diagnosed at a late stage. Detection at an earlier stage can dramatically improve prognosis. Microscopic evaluation of esophageal balloon cytology (EBC) specimens has been the most common method for early detection of ESCC, but this technique is limited by low sensitivity and specificity. The use of molecular markers may improve these screening characteristics. This study evaluates whether measurement of gene methylation in EBC specimens may have utility for the detection of esophageal squamous dysplasia and early ESCC. We evaluated the presence of methylation in eight genes shown to be methylated in ESCC in previous studies in EBC specimens from 147 patients with endoscopic biopsy diagnoses ranging from normal mucosa to severe squamous dysplasia. Methylation status was determined using quantitative methylation-specific PCR techniques. The sensitivity and specificity of methylation of each individual gene and of combinations of these genes to detect biopsyproven high-grade (moderate or severe) squamous dysplasia were determined. For individual genes, the sensitivities ranged from 9% to 34% and the specificities ranged from 77% to 99%. Using a panel of four genes (AHRR, p16INK4a, MT1G, and CLDN3) resulted in sensitivity and specificity of 50% and 68%, respectively. This study suggests that evaluation of gene methylation in EBC samples may have utility for early detection of esophageal squamous dysplasia and early ESCC; however, identification of more sensitive methylation markers will be required for development of a clinically useful screening test.

Topics: Adult; Aged; Barrett Esophagus; Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; Carcinoma, Squamous Cell; Claudin-3; DNA Methylation; Early Detection of Cancer; Esophageal Neoplasms; Esophagoscopy; Female; Genes, p16; Humans; Male; Membrane Proteins; Metallothionein; Middle Aged; Precancerous Conditions; Promoter Regions, Genetic; Repressor Proteins; Sensitivity and Specificity

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, inducible, intracellular proteins that bind heavy metals with high affinity. MT-1 is known as a stress-inducible protein and functions as an antioxidant enzyme. Areca quid chewing is a major risk factor in the development and further progression of oral squamous cell carcinoma (OSCC). The aim of this study was to compare MT-1 expression in normal human oral epithelium and OSCC and further explore the potential mechanism that may lead to induce MT-1 expression. Thirty four OSCC and 10 normal epithelium specimens were examined by immunohistochemistry and analyzed by the clinico-pathological profiles. The oral epithelial cell line GMN cells were challenged with arecoline, a major areca nut alkaloid, by reverse-transcriptase polymerase chain reaction. Furthermore, tobacco smoke carcinogen benzo[a]pyrene (BaP) and glutathione (GSH) precursor N-acetyl-l-cysteine (NAC) were added to find the possible regulatory mechanisms. The results from immunohistochemistry demonstrated that MT-1 expression was significantly higher in OSCC specimens (p<0.05). No significant difference in MT-1 expression was observed with respect to age, sex, T category, and stage (p>0.05). The high MT-1 expression was associated with lymph node metastasis (p=0.012). In addition, arecoline was found to elevate MT-1 mRNA in a dose-dependent manner (p<0.05). Furthermore, the addition of BaP enhanced the arecoline-induced MT-1 expression (p<0.05). The addition of NAC markedly inhibited the arecoline-induced MT-1 expression (p<0.05). These results lead to the conclusion that MT-1 expression is significantly upregulated in areca quid chewing associated-OSCC. The expression profile suggests MT-1 could be used clinically as a marker for tumors possessing the potential for lymph node metastasis. The compounds of tobacco products may act synergistically in the pathogenesis of OSCC in areca quid chewers. The regulation of MT-1 expression induced by arecoline is critically dependent on the intracellular GSH concentration.

Topics: Adult; Aged; Areca; Carcinoma, Squamous Cell; Case-Control Studies; Chi-Square Distribution; Female; Humans; Immunohistochemistry; Male; Metallothionein; Middle Aged; Mouth Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Up-Regulation

Esophageal squamous cell carcinoma (ESCC) in the Indian population is associated with poor nutritional status, low socioeconomic conditions, bidi smoking and consumption of smokeless tobacco products, besides alcohol drinking and cigarette smoking. To determine the impact of these risk factors on molecular pathogenesis of ESCC, we determined global gene expression profiles of 7 paired samples of ESCC and histologically confirmed nonmalignant esophageal tissues using 19.1K cDNA microarrays. The most salient finding was identification of 19 differentially expressed genes encoding zinc binding or modulating proteins associated with transcriptional regulation, ubiquitin-protein degradation and maintenance of zinc homeostasis. Validation of differential expression of a subset of genes by real-time quantitative RT-PCR (real-time QRT-PCR) in clinical specimens of ESCC, esophageal dysplasia and histologically nonmalignant esophageal tissues and immunohistochemical analysis using tissue microarrays confirmed the microarray data and demonstrated upregulation of zinc finger proteins, cellular modulator of immune recognition (c-MIR), snail homolog 2 (SLUG), zinc transporter, ZnT7 and downregulation of zinc metabolizing protein, metallothionein MT1G. We also observed upregulation of mitogen activated protein kinase kinase kinase 3 (MAP3K3/MEKK3), a kinase anchor protein 13 (AKAP13) and transglutaminase2 (TG2). Interestingly, we found upregulation of ZnT7 transcripts in ESCC cells (TE13) grown in zinc deficient condition. In conclusion, our data suggest deregulation of genes associated with zinc homeostasis in ESCC.

Topics: Carcinoma, Squamous Cell; Cation Transport Proteins; Esophageal Neoplasms; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Homeostasis; Humans; Immunohistochemistry; Male; Metallothionein; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; RNA, Neoplasm; Tumor Cells, Cultured; Zinc; Zinc Fingers

We examined metallothionein (MT) expression in bladder cancer and its relationship to clinicopathologic factors, survival data, and outcome of chemotherapy. In 97 patients who underwent radical cystectomy for bladder cancer, 34 of whom received cisplatin-based chemotherapy, MT expression was evaluated immunohistochemically. Results were correlated with histopathologic data, survival rates, and outcome of chemotherapy. MT overexpression was present in 33 patients (34.0%): strong in 7 (7.2%) and focal in 26 (26.8%). Overexpression was an independent prognostic factor and was significantly associated with poor survival. Patients undergoing chemotherapy showed worse survival if their tumours were MT-positive than if they were MT-negative. MT overexpression predicts unfavorable survival in bladder cancer patients. In those treated with cisplatin chemotherapy, survival is significantly poorer if tumours express MT. Our results show that MT overexpression may mediate resistance to alkylating agents. Therefore, further studies are warranted to define those patients who need a more aggressive therapy.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cisplatin; Female; Humans; Male; Metallothionein; Middle Aged; Retrospective Studies; Survival Rate; Treatment Outcome; Urinary Bladder Neoplasms

Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most frequent skin cancer. Its pathogeny is linked to genotoxic effects of actinic radiation exposure, especially to ultraviolet wavelength. Metallothionein (MT) is a low-molecular weight protein with high affinity for heavy metal. Its intracellular function has been related to heavy metals and free-radical detoxification, although many studies linked MT to protective action against actinic mutagenesis. In other way, overexpression in malignant tumors has been related to worse prognosis. We aimed to evaluate MT immunohistochemical expression in skin cancer associated to actinic radiation. Twenty-six BCC cases, 20 SCC, and 6 normal skin fragments were investigated. Immunohistochemical assay were performed by streptavidin-biotin-peroxidase technique with standard monoclonal antibody (E9). In normal skin, immunostaining was observed in basal layer of the epithelium. In the epithelium adjacent to tumors, suprabasal layer was also intensely labeled. Mean MT immunostaining indices were 18.5+21.2% for BCC and 69.1+14.4% for SCC. This difference was statistically significant. Higher MT expression in SCC as compared with BCC suggests association with tumoral aggressiveness.

Topics: Aged; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Female; Humans; Immunohistochemistry; Male; Metallothionein; Skin Neoplasms; Ultraviolet Rays

Esophageal squamous cell carcinoma (ESCC) is a common cancer with a very poor prognosis. New methods are needed to screen high-risk populations and identify curable tumors and precursor lesions early. Molecular markers may be useful in such screening efforts. This study was designed to determine the prevalence of p16, MGMT, RARbeta2, CLDN3, CRBP and MT1G gene methylation in patients with ESCC to evaluate the variation of gene methylation across a spectrum of preneoplastic lesions, and assess the feasibility of using gene methylation in a primary screening test utilizing frozen esophageal cells collected by balloon cytology samplers. Samples were obtained from high-risk subjects from north central China. These samples included 11 foci of histologically normal mucosa, 8 foci of low-grade squamous dysplasia, 7 foci of high-grade squamous dysplasia, and 13 foci of ESCC from 6 fully embedded resection specimens; endoscopic biopsies from 6 individuals with no histological evidence of disease; and frozen esophageal balloon samples from 12 asymptomatic subjects. Promoter CpG site-specific hypermethylation status was determined for each gene using real-time methylation-specific PCR (qMS-PCR) based on Taqman chemistry. Of the 6 ESCC patients, 5 showed methylation of at least one gene. For most genes, methylation occurred with increasing frequency during neoplastic progression, with the largest increase found between low- and high-grade dysplasia. There was considerable variation in methylation patterns among different foci of the same histological grade, even within individual patients, but 16/20 (80%) of high-grade dysplastic and cancer foci had >or= 2 methylated genes, while 17/19 (89%) of normal and low-grade dysplastic foci had <2 methylated genes. These genes were rarely methylated in histologically normal mucosa from patients with or without ESCC. Gene methylation was common and easily detectable in the frozen esophageal cells collected by balloon cytology samplers. Our data suggest that methylation of p16, MGMT, RARbeta2, CLDN3, CRBP, and MT1G is common in the esophageal mucosa of patients with ESCC in this high-risk population, and tends to increase in prevalence in foci with increasing histological severity of disease. Methylation data from panels of genes may be able to identify patients with high-grade lesions. Balloon cytology may be able to screen the length of the esophagus effectively for a subset of cells with abnormal methylation, and may be useful

Topics: Adult; Aged; Carcinoma, Squamous Cell; Claudin-3; DNA Methylation; DNA Modification Methylases; DNA Repair Enzymes; Esophageal Neoplasms; Female; Genes, p16; Humans; Male; Membrane Proteins; Metallothionein; Middle Aged; Precancerous Conditions; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Tumor Suppressor Protein p14ARF; Tumor Suppressor Proteins

Zinc deficiency in rats enhances esophageal cell proliferation, causes alteration in gene expression, and promotes esophageal carcinogenesis. Zinc replenishment rapidly induces apoptosis in the esophageal epithelium thereby reversing cell proliferation and carcinogenesis. To identify zinc-responsive genes responsible for these divergent effects, we did oligonucleotide array-based gene expression profiling analyses in the precancerous zinc-deficient esophagus and in zinc-replenished esophagi after treatment with intragastric zinc compared with zinc-sufficient esophagi. Thirty-three genes (21 up-regulated and 12 down-regulated) showed a > or = 2-fold change in expression in the hyperplastic zinc-deficient versus zinc-sufficient esophageal epithelia. Expression of genes involved in cell division, survival, adhesion, and tumorigenesis were markedly changed. The zinc-sensitive gene metallothionein-1 (MT-1 was up-regulated 7-fold, the opposite of results for small intestine and liver under zinc-deficient conditions. Keratin 14 (KRT14, a biomarker in esophageal tumorigenesis), carbonic anhydrase II (CAII, a regulator of acid-base homeostasis), and cyclin B were up-regulated >4-fold. Immunohistochemistry showed that metallothionein and keratin 14 proteins were overexpressed in zinc-deficient esophagus, as well as in lingual and esophageal squamous cell carcinoma from carcinogen-treated rats, emphasizing their roles in carcinogenesis. Calponin 1 (CNN1, an actin cross-linking regulator) was down-regulated 0.2-fold. Within hours after oral zinc treatment, the abnormal expression of 29 of 33 genes returned to near zinc-sufficient levels, accompanied by reversal of the precancerous phenotype. Thus, we have identified new molecular markers in precancerous esophagus and showed their restoration by zinc replenishment, providing insights into the interaction between zinc and gene expression in esophageal cancer development and prevention.

Topics: 4-Nitroquinoline-1-oxide; Animals; Carcinoma, Squamous Cell; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Immunohistochemistry; Keratin-14; Keratins; Male; Metallothionein; Precancerous Conditions; Rats; Reverse Transcriptase Polymerase Chain Reaction; Up-Regulation; Zinc

Esophageal squamous cell carcinoma is generally sensitive to chemoradiotherapy (CRT), but some cases are not. Using a retrospective analysis, we aimed to identify the predictors of the response by esophageal squamous cell carcinoma to definitive CRT.. The intensities of expression of p53, Ki67, Bcl-2, Bax, cyclin D1, VEGF, CDC25B, and metallothionein (MT) were evaluated immunohistochemically in the biopsy specimens obtained before CRT, and the intensities of their expression were tested for correlations with the clinical effects of CRT.. The esophageal squamous cell carcinomas with negative p53, positive CDC25B, and negative MT expression were found to be significantly more sensitive to CRT. In addition, p53 positivity and CDC25B positivity respond well to CRT.. Esophageal squamous cell carcinomas with negative p53, positive CDC25B, and negative MT expressions respond well to CRT. Even with p53 positivity, if with CDC25B positivity, CRT can be expected.

Topics: Adult; Aged; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Squamous Cell; cdc25 Phosphatases; Cell Cycle Proteins; Esophageal Neoplasms; Female; Humans; Male; Metallothionein; Middle Aged; Retrospective Studies; Tumor Suppressor Protein p53

An accumulation of genetic alterations forming the field of cancerization is an important event for the transformation from normal to cancer cell in multistep carcinogenesis. Histopathologically healthy tumor adjacent tissue might be considered as a cancerization field which is typified by genetic changes required for the development of cancer. Metallothionein (MT) is considered to be a protective and anti-apoptotic protein. The aim of our study was to evaluate the MT expression in head and neck squamous cells carcinoma and breast adenocarcinoma and their histologically healthy adjacent tissue.. We have sampled 29 tissue samples in total derived from head and neck cancers and 29 samples of their clear surgical margins, 33 breast adenocarcinomas and 33 clear surgical margins. Antibody recognizing MT-1 was used for immunohistochemical analysis.. MT expression was revealed in 85,7% of head and neck cancers and 94% of breast adenocarcinomas. It was found in all tumor adjacent tissue. MT expression was statistically significantly higher in tumor adjacent tissue than in cancer tissue in cases with the presence of lymph node metastases in both, breast adenocarcinoma and head and neck squamous cell carcinoma. Generally stroma seems to respond to the presence of cancer by the expression of MT, even in tissues which normally do not express MT.. MT might be a normal or protective reaction of healthy adjacent tissue to the presence of tumor.

Topics: Adenocarcinoma; Adult; Aged; Breast Neoplasms; Carcinoma, Squamous Cell; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; Metallothionein; Middle Aged; Stromal Cells

Metallothionein (MT) is a low molecular weight protein, which participates in differentiation and proliferation of normal and tumour cells. In some malignant tumours (mammary, renal, ovarian cancers), its increased expression is thought to represent an unfavourable prognostic factor. Non-small-cellular lung cancers (mainly squamocellular cancer and adenocarcinoma) are characterised by ill-defined prognosis, which poses problems in the selection of effective post-surgical therapy. The present study aimed at demonstration of the prognostic significance of MT expression in cells of non-small cell lung cancers, attempting to correlate the intensity of MT expression with G grade and with the intensity of proliferation-associated antigen, Ki-67 expression. The studies were performed on archival paraffin blocks with samples of 25 cases of non-small cell lung cancers (5 squamous cell cancers, 20 adenocarcinomas). In paraffin sections of the studied tumours, immunocytochemical reactions were performed, using mouse monoclonal anti-MT and anti-Ki-67 antibodies. The expressions of MT and Ki-67 were demonstrated in all the studied tumours. An analysis of correlation between the expression of MT, Ki-67 antigen and G grade demonstrated a strong positive relation between the latter two parameters (r=0.70; p<0.05). Less pronounced positive correlations were disclosed between MT expression and G grade (r=0.44; p<0.05) and between MT expression and the expression of Ki-67 antigen (r=0.41; p<0.05). In addition, in 15 cases of examined tumours, survival analysis was performed, which disclosed a shorter survival in patients with high MT expression. The obtained results confirmed the relationship between MT expression and Ki-67 antigen expression, indicating an involvement of the proteins in processes of tumour cell proliferation. In turn, the shorter survival of patients with high expression of MT pointed to prognostic significance of the protein in non-small cell lung cancers.

Topics: Adenocarcinoma; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Humans; Ki-67 Antigen; Lung Neoplasms; Metallothionein; Survival Analysis

The purpose of the study was twofold: 1) to search for potential biomarkers that were overexpressed in cell lines that could represent both a clinical premalignant (immortalized) and a malignant state, and 2) to attempt to correlate metallothionein gene expression with clinical outcome in laryngeal carcinoma.. A series of in vitro experiments were used to unearth differentially expressed genes among normal, immortalized and tumorigenic cell lines. Secondarily, a retrospective analysis was undertaken.. Differential display analysis was conducted to identify differentially expressed genes between human papillomavirus-infected immortalized HOK16B and benzo[ ]pyrene-derived tumorigenic cell line, HOK16B-BaP-T. The cell-specific expressions were examined by Northern blot analysis and compared with other known immortalized and cancer cell lines. Immunohistochemical staining was also conducted to localize metallothionein (MT I/II) protein expression among the different cell lines studied. A retrospective analysis of laryngeal specimens from archival tissues of 29 cancer patients who underwent primary surgical resection was also undertaken after immunohistochemical staining.. Twenty-one differentially expressed complementary cDNA clones, both novel and known, were identified using the differential display analysis. Northern blot analysis confirmed that clone 6 hybridized to a 1.6-kb RNA in HOK16B-Bap-T cell line. Clone 4 showed decreased expression in immortalized and cancer cell compared with NHOK. MT I/II transcript was observed in HOK16B, which was further elevated in HOK16B-Bap-T. Retrospective analysis showed that high immunoreactivity to MT I/II in surgically resected laryngeal cancer specimen correlated with increased frequency of recurrence within 2 years of surgery.. These findings suggest that clone 4 may potentially function as a tumor suppressor gene, which may be significant in tumor progression and invasion. Clone 6 may participate in viral-mediated oncogenic transformation of normal cells. Clone 6 may also have potential as a tumor maker differentiating normal from malignant tissue, as in the determination of surgical resection margins. MT I/II gene product may serve as a prognostic biomarker for laryngeal squamous cell carcinoma. The differentially expressed genes and gene products may serve as sensitive biomarkers for improved early detection, diagnosis, and prognosis of head and neck squamous cell carcinoma.

Topics: Bacterial Proteins; Bacterial Toxins; Blotting, Northern; Carcinoma, Squamous Cell; Cell Culture Techniques; Clone Cells; DNA, Complementary; Escherichia coli Proteins; Female; Gene Expression; Genes, Tumor Suppressor; Humans; Immunohistochemistry; Laryngeal Neoplasms; Male; Metallothionein; Middle Aged; Neoplasm Staging; Retrospective Studies; RNA

Pimonidazole binding (hypoxia) and involucrin expression (differentiation) overlap extensively in squamous cell carcinomas. This study asks whether involucrin might serve as an endogenous marker for tumor hypoxia. A second question is whether differentiation affects hypoxia-inducible metallothionein (MT) expression in normal human epithelia and squamous cell carcinomas as it does in rodent epithelia.. Thirty-four patients with squamous cell carcinoma of the uterine cervix were infused with pimonidazole hydrochloride solution. The next day, multiple biopsies were formalin-fixed, paraffin-embedded and sectioned at 4 micro m. Qualitative and quantitative analyses for involucrin expression, pimonidazole binding, and human MT-IIa mRNA expression were performed.. No overall correlation between the extent of involucrin expression and pimonidazole binding was observed. The lack of correlation was because of heterogeneous patterns of immunostaining for involucrin generally related to tumor grade. Colocalized immunostaining for involucrin and pimonidazole binding was observed in intermediate grade tumors but not in well-differentiated or poorly differentiated tumors. Human MT-IIa mRNA and MT protein were expressed in basal lamina of normal human epithelia and in the proliferative rims of tumor nests.. Colocalization of immunostaining for involucrin and pimonidazole binding is consistent with oxygen regulation, but the lack of involucrin expression in hypoxic regions of poorly differentiated tumors indicates that its transcriptional status with respect to hypoxia induction is altered by cell differentiation. The localization of MT message and protein in the outer rims of most tumor nests indicates that the transcriptional status of metallothionein is also altered by differentiation.

Topics: Biopsy; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Female; Humans; Hypoxia; Immunohistochemistry; In Situ Hybridization; Metallothionein; Nitroimidazoles; Oxygen; Prognosis; Protein Precursors; Radiation-Sensitizing Agents; RNA, Messenger; Transcription, Genetic; Uterine Cervical Neoplasms

The aim of the study was to determine the contribution of metallothionein (MT) and p53 expression in predicting laryngeal squamous cell carcinoma (SCC) recurrence. This was a retrospective study in which MT and p53 immunopositive staining in 32 laryngeal SCC paraffin-embedded sections, were correlated with clinical recurrence. Recurrence was observed in 8 cases with MT expression (42.1%) and 1 case with no expression (7.7%). Moderate and strong MT expression was associated with 14.3% and 58.3% recurrence, respectively. Recurrence was similar for both p53-negative (21.1%) and p53-positive (27.3%) groups. One third of the patients expressing both p53 and MT simultaneously had recurrence. Thus, the combined expression of p53 and MT did not improve the predictive value for recurrence compared to MT alone. MT over-expression may be an independent risk factor for laryngeal SCC recurrence.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Metallothionein; Middle Aged; Neoplasm Recurrence, Local; Prognosis; Tumor Suppressor Protein p53

Metallothionein (MT), a low-molecular-weight protein with high cysteine content, seems to be related to neoplastic resistance to oncologic treatment and therefore has been studied as a prognostic factor for a variety of human malignant tumors. MT overexpression in neoplasms of ectodermal origin is usually associated with a poor prognosis. MT expression was evaluated in 60 samples of oral squamous cell carcinoma by immunohistochemistry to study its prognostic influence on oral cancer. Possible associations of MT immunoexpression were also investigated with respect to clinical stage (TNM), histological grading, and proliferation index (Ki-67) of the lesions. No significant statistical correlation was observed among these variables. The impact on overall survival was assessed by uni and multivariate statistical tests. Mean MT labeling index was 60%. High MT labeling indexes (over 76%) predicted shorter survival in univariate statistical analysis. In multivariate analysis, MT labeling index and clinical stage were independent prognostic factors. MT overexpression in oral squamous cell carcinoma seems to be related to a worse prognosis for patients.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cell Division; Female; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Male; Metallothionein; Middle Aged; Mouth Neoplasms; Neoplasm Staging; Survival Analysis; Survival Rate

Over-expression of cellular metallothionein occurs frequently in human tumours but the underlying mechanism remains unknown. The aim of this study was to assess metallothionein expression in cases of lung carcinoma and to correlate it with histopathological parameters.. Tumour tissue samples from 89 patients with lung carcinoma were immunostained by the streptavidin-biotin-peroxidase technique, using a monoclonal antibody against both metallothionein-1 and -2 isoforms. Positive matallothionein immunostaining was prominent in 44 out of 89 (49%) and negative in 45 out of 89 (51%) cases of lung carcinoma examined. Metallothionein positivity was prominent in 32 out of 43 (74%) cases of squamous cell lung carcinoma, and in 12 out of 35 (34%) cases of adenocarcinoma, while it was negative in all 11 cases of small-cell lung carcinoma examined, presenting a statistically significant difference between the different histological types. The intensity of metallothionein staining revealed a statistically significant difference between the squamous cell and adenocarcinoma cases examined. The pattern and extent of metallothionein staining in tumour cells and the expression of metallothionein in stromal cells were not correlated with histopathological parameters (type and grade) in metallothionein-positive cases of lung carcinoma examined. No association was found between metallothionein expression and lymph node status in the examined cases of lung carcinoma.. Our findings indicate that expression of metallothionein was evident in squamous cell lung carcinoma and adenocarcinoma, but absent in small-cell lung carcinoma, supporting evidence for participation of this protein in the biological mechanisms underlying the carcinogenic evolution in the lung.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Female; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Metallothionein; Middle Aged

The sensitivity of cancer cells to chemotherapy and radiation therapy depends on various biological properties. This study investigated the expression of p53, CDC25B and metallothionein (MT), and evaluated their clinical significance in chemoradiation therapy (CRT) for oesophageal squamous cell carcinoma.. The expression of p53, CDC25B and MT was evaluated by immunohistochemistry using biopsy specimens taken before CRT for 77 patients with oesophageal squamous cell carcinoma, and correlated with the pathological effects of CRT and survival.. p53-positive tumours and MT-positive tumours had a poor response to CRT, whereas tumours with strong CDC25B expression were associated with a good response. When each patient was scored for the presence of the three biological factors, there was a strong correlation between the sensitivity score and the pathological effect of CRT (P < 0.001), and a (non-significant) difference in the 5-year survival rate between patients with a high score and those with a low score (67 versus 34 per cent respectively; P = 0.12).. The combined evaluation of p53, CDC25B and MT may help to identify patients with advanced oesophageal squamous cell carcinoma who will benefit from preoperative CRT.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; cdc25 Phosphatases; Cell Cycle Proteins; Cisplatin; Esophageal Neoplasms; Female; Fluorouracil; Genes, p53; Humans; Immunohistochemistry; Infusions, Intravenous; Male; Metallothionein; Middle Aged; Survival Analysis; Treatment Outcome

The purpose of this study was to define the prognostic value of a group of molecular tumor markers in a well-staged population of patients treated with trimodality therapy for esophageal cancer. The original pretreatment paraffin-embedded endoscopic esophageal tumor biopsy material was obtained from 118 patients treated with concurrent cisplatin + 5-fluorouracil (5-FU) + 45 Gy radiation followed by resection from 1986 until 1997 at the Duke University Comprehensive Cancer Center. Three markers of possible platinum chemotherapy association [metallothionein (MT), glutathione S-transferase-pi (GST-pi), P-glycoprotein (P-gp or multidrug resistance)] and one marker of possible 5-FU association [thymidylate synthase (TS)] were measured using immunohistochemistry. The median cancer-free survival was 25.0 months, with a significantly improved survival for the 38 patients who had a complete response (P < 0.001). High-level expression of GST-pi, P-gp, and TS were associated with a decreased survival. MT was not significant in this population. Multivariate analysis identified high-level expression in two of the platinum markers (GST-pi and P-gp) and the 5-FU marker TS as independent predictors of early recurrence and death. In conclusion, this investigation measured three possible markers associated with platinum and one possible marker associated with 5-FU in a cohort of esophageal cancer patients. Independent prognostic significance was observed, which suggests that it may be possible to predict which patients may benefit most from trimodality therapy. These data need to be reproduced in a prospective investigation.

Topics: Adenocarcinoma; Adult; Age Factors; Aged; Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biopsy; Carcinoma, Squamous Cell; Cisplatin; Disease-Free Survival; Esophageal Neoplasms; Fluorouracil; Glutathione S-Transferase pi; Glutathione Transferase; Humans; Immunohistochemistry; Isoenzymes; Metallothionein; Middle Aged; Multivariate Analysis; Prognosis; Proportional Hazards Models; Thymidylate Synthase; Time Factors; Treatment Outcome

We analyzed 53 specimens from primary squamous cell carcinomas of the skin for the expression of collagenase IV, cathepsin D and metallothionein by means of immunohistochemistry along with histological data. We compared tumors that metastasized (30) with tumors that did not (23) during a follow-up period of at least 5 years. The expression of the two proteolytic enzymes cathepsin D and collagenase IV was also assessed at the invading front of the tumors. Statistical analyses revealed significant differences for the overall expression of cathepsin D (P < 0.05), expression of cathepsin at the invading front (P < 0.05) and the tumor thickness (P < 0.01). The results showed that cathepsin D may be a prognostic factor for squamous cell carcinomas of the skin. We then combined the results of parameters with statistically significant differences by multiplication. The prognostic value of such a combined risk-factor score showed higher confidence than any of the single parameters alone: a sensitivity of 63.3%, a specificity of 87.0% and an efficiency of 73.6%. This kind of combined risk-factor score might be used to more accurately detect patients at high risk of metastasis.

Topics: Aged; Carcinoma, Squamous Cell; Cathepsin D; Female; Humans; Immunohistochemistry; Male; Matrix Metalloproteinase 2; Metallothionein; Predictive Value of Tests; Prognosis; Skin Neoplasms

The goal of this study is to clarify whether the expression of metallothionein (MT) could affect the prognosis and the metastatic potential of squamous cell carcinoma (SCC) of the oesophagus. In paraffin-embedded specimens resected from 57 patients, MT mRNA and protein expressions were detected by in situ hybridization and immunohistochemistry respectively. The expression of MT was evaluated in respect of clinicopathologic variables and patients' survival. MT mRNA expression was significantly associated with the proportion of lymph node metastasis (71% in MT mRNA-positive tumours vs 42% in MT mRNA-negative tumours; P = 0.0343) and that of distant metastasis (29% in MT mRNA-positive tumours vs 5% in MT mRNA-negative tumours; P = 0.0452). In respect of MT protein expression, the frequency of distant metastasis was more common in MT-positive tumours than in MT-negative tumours (30% in MT-positive tumours vs 8% in MT-negative tumours; P = 0.0446). The survival rate of the patients with MT protein-negative tumours was significantly better than that of the patients with MT protein-positive tumours (P = 0.0340). There was a positive correlation between the expression of MT protein and that of proliferating cell nuclear antigen (P = 0.0018). Therefore, we conclude that MT expression, both at the mRNA and protein levels, may be a potential marker predicting metastatic and proliferative activities of oesophageal SCC.

Topics: Aged; Base Sequence; Carcinoma, Squamous Cell; Cell Division; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; In Situ Hybridization; Male; Metallothionein; Middle Aged; Molecular Sequence Data; Proliferating Cell Nuclear Antigen; RNA, Messenger

In this study we evaluated the immunohistochemical expression of metallothionein (MT) in 44 squamous cell carcinomas, 14 cases of in situ carcinoma, 47 with epithelial dysplasia, 11 papillomas and 21 cases of keratosis. The MT expression was studied in correlation with p53 protein expression and the proliferative cell nuclear antigen (PCNA). The monoclonal antibodies E9 (anti-MT), DO-7 (which reacts with a denaturation-resistant epitope in wild-type and mutant p53) and PC10 (anti-PCNA) on formalin-fixed, paraffin-embedded tissue were used employing the immunoperoxidase (ABC) method. The immunohistochemical localization of MT has shown its rather ubiquitous presence in the cytoplasm and nucleus of both benign and malignant epithelial cells. In most cases the adjacent "normal" epithelium showed low positivity in the basal portion. The mean value of metallothionein expression was 35.73 in squamous cell carcinomas, 32.21 in in situ carcinomas, 11.86 in dysplastic epithelium, 5.10 in papillomas and 3.5 in keratosis. In carcinomas, low MT expression (< 10% of neoplastic cells) was observed in 20.5% of the cases, moderate (10%-50% of neoplastic cells) in 54.5% and extensive expression (> 50% of neoplastic cells) in 25% of the cases. We did not find any statistically significant difference of MT expression between in situ and infiltrating carcinomas, while we did observe a significant difference between carcinomas and the other groups. There was a statistically significant difference in the PCNA values in both benign and malignant lesions, while no statistically significant difference was observed in p53 protein expression in the above groups. A positive correlation between MT expression and the PCNA value (p < 0.0001) in the benign and malignant groups was detected. The PCNA value was also correlated with the p53 protein expression (p = 0.001). No correlation was found between MT and p53 protein expression. In conclusion, these results suggest that the MT expression may play a role in the development of malignant disease of the larynx, from the early phase of laryngeal carcinogenesis, independently from the p53 expression. It is also possible to contribute to tumour cell growth, as determined by the PCNA score.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma in Situ; Carcinoma, Squamous Cell; Epithelial Cells; Female; Humans; Immunoenzyme Techniques; Keratosis; Laryngeal Neoplasms; Male; Metallothionein; Middle Aged; Papilloma; Precancerous Conditions; Proliferating Cell Nuclear Antigen; Tumor Suppressor Protein p53

Metallothioneins (MTs) are ubiquitous low molecular weight proteins with a high affinity for heavy metal ions such as zinc, copper, cadmium, and platinum. Immunohistochemically detectable MT overexpression has been demonstrated in a variety of cancers, especially breast carcinoma. In this study, the immunohistochemical expression of MT in normal cervical squamous epithelia, cervical intraepithelial neoplasms (CINs), and invasive cervical squamous carcinomas was investigated. Immunohistochemical staining for proliferating cells using the MIB1 antibody was also performed. In normal squamous epithelia (n = 31), positive staining with MT was confined to basal and parabasal cells. In cases of koilocytosis (n = 14) and CIN I (n = 10), staining was also largely confined to basal and parabasal cells, with only occasional cases of CIN I exhibiting positivity within higher cell layers. Cases of CIN II (n = 14) showed positive staining largely confined to basal and parabasal cells, with staining of higher cell layers in a few cases. In the majority of cases of CIN III (n = 29), there was diffuse positive staining throughout the full epithelial thickness and, in almost all cases, positive staining was present above the basal and parabasal layers. Positive staining was present in 19 of 21 invasive squamous carcinomas. With MIB 1, positivity was confined to the parabasal layer in normal squamous epithelia. In cases of CIN, positive cells were present in progressively higher cell layers, in accordance with the grade of CIN. There was widespread positive staining in all cases of invasive squamous carcinoma. Overexpression of MT, demonstrated immunohistochemically, is associated with CIN III and invasive cervical squamous carcinoma, lesions which exhibit the highest proliferative activity, as shown by MIB1 immunostaining. MT overexpression in cervical squamous lesions appears to occur at some point along the spectrum of high grade CIN and may be related to cell proliferation.

Topics: Antigens, Nuclear; Autoantigens; Carcinoma, Squamous Cell; Cervix Uteri; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Metallothionein; Nuclear Proteins; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

Cells with acquired anticancer drug resistance frequently exhibited broad cross-resistance to other anticancer agents. Increased human metallothionein (hMT) IIa transcription has been found in some cells with acquired resistance to cisplatin (CP). A panel of 5'hMT-IIa promoter deletions linked to the chloramphenicol acetyl transferase ((5'-hMT-IIaCAT) were used to demonstrate that certain cis-elements are important for the increased hMT-IIa transcription in CP-resistant cells (SCC25/CP) compared to CP-sensitive cells (SCC25). We further identified trans-acting factor differences between SCC25 and SCC25/CP cells using gel mobility shift assays. Significant increases in binding of specific factors to the -286 to -160 and -96 to -51 region were seen in CP-resistant SCC25/CP cells compared to sensitive SCC25 cells. Using cross-competition and super gel shift analyses, we identified enhanced Spl and AP-2 binding activity in SCC25/CP cells. By Western blot analysis and immunoprecipitation, we demonstrated that the level of Spl was unchanged between the two cells types whereas AP-2 was elevated twofold in SCC25/CP cells. Our results indicated that the selection of CP-resistant phenotype in SCC25/CP was accompanied by increased Spl and AP-2 DNA binding activities, which are likely not only to enhance hMT-IIa transcription but could also alter expression of other genes responsible for a broader anticancer drug cross-resistance. Thus, altered trans-acting factors could account for the complex cross-resistant phenotype found in some anticancer drug-resistant cells.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Nucleus; Chloramphenicol O-Acetyltransferase; Cisplatin; Drug Resistance, Neoplasm; Electrophoresis, Agar Gel; Genes, Reporter; Genetic Vectors; Head and Neck Neoplasms; Humans; Metallothionein; Neoplasm Proteins; Promoter Regions, Genetic; Transcription, Genetic; Transcriptional Activation; Transfection; Tumor Cells, Cultured

The Fas receptor appears to be commonly expressed in all morphological types of epithelial laryngeal hyperplasia (HP). Fas-mediated apoptotic cell death would thus be a possible phenomenon in these lesions. We observed more anti-apoptotic bcl-2 protein in epithelia with simple HP compared to the more advanced types of HP. It is suggested that in simple HP there is not yet a need for an early selection for cell death. The observed overexpression of metallothionein (MT) in the basal layers of simple HP would also support such a theory. These basal cells are dividing, non-apoptotic cells, which have not yet been selected for death. All 20 cysteine residues in MT are involved in metal binding, interfering with the intracellular redox balance, and thereby possibly inhibiting certain apoptotic signals. MIB-1 positivity was found only in the atypical HP, CIS, and invasive carcinomas. Intuition suggests that high labelling would be associated with poor prognosis. The degree of apoptosis, evaluated by TUNEL, did not show any differences between different types of epithelia. Although TUNEL is sensitive and rather specific, we emphasise that all TUNEL positive cells have apoptotic type morphology, confirming good and appropriate use of the technique.

Topics: Apoptosis; Carcinoma in Situ; Carcinoma, Squamous Cell; Epithelium; Fas Ligand Protein; Humans; Hyperplasia; Laryngeal Diseases; Laryngeal Mucosa; Laryngeal Neoplasms; Membrane Glycoproteins; Metallothionein; Neoplasm Invasiveness; Precancerous Conditions; Proto-Oncogene Proteins c-bcl-2

Metallothionein (MT) is a chelator present in myoepithelial cells, whilst the Fas-receptor (APO-1, CD95) has been described primarily in human T Jurkat cells. 20 cases of carcinoma of the tongue were investigated immunocytochemically with regard to MT, Fas and Bcl-2. In normal oral squamous epithelium, MT is located in the basal/parabasal dividing cells only. In well-differentiated nests of carcinomas, MT is observed almost entirely in peripherally located cells. In situ end-labelling indicates apoptosis in the centre of these nests, but not in the peripheral areas. Less-differentiated areas show more general MT-positivity, but little apoptosis. All 24 tumours are Fas-positive, but normal epithelia are mainly negative (P < 0.0001). Bcl-2 protein was sparse in the tumours compared with MT and Fas (P < 0.0001). We thus suggest that MT, possibly due to its chelating properties, may contribute to delaying cells entering apoptosis, both in normal epithelium near the base and in less-differentiated regions of carcinoma. Moreover, Fas may be present in cells of human malignancies, as well as those of established malignant cell lines.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Differentiation; fas Receptor; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Metallothionein; Middle Aged; Tongue Neoplasms

Topics: Animals; Antibodies; Blotting, Western; Carcinoma, Squamous Cell; Carrier Proteins; Cell Line; Electrophoresis, Polyacrylamide Gel; Humans; Liver; Metallothionein; Rabbits; Tumor Cells, Cultured

This study was undertaken to elucidate the mechanism(s) of potentiation of cisplatin (CDDP) cytotoxicity by interferon alpha-2a (IFN alpha-2a) in human squamous carcinoma cell lines SCC-25 and SCC-4. IFN alpha-2a treatment significantly increased the cytotoxicity of CDDP in both cell lines in a dose-dependent manner. In SCC-25 cells, the cytotoxicity of CDDP was increased by about 2- and 4-fold, respectively, by treating the cells with 400 and 800 IU/ml IFN alpha-2a. Sensitivity of SCC-4 cells to CDDP was increased by about 3- and 7-fold, respectively, by 400 and 800 IU/ml IFN alpha-2a treatment. Drug uptake experiments revealed approximately 1.4- to 5-fold higher platinum accumulation in IFN alpha-2a-treated cells as compared to respective controls. Cellular levels of glutathione (GSH) and GSH transferase, which have been suggested to be important determinants of tumor cell sensitivity to CDDP, were not altered by IFN alpha-2a treatment in either of the cell lines. Northern blot analysis showed a moderate increase (about 30-40%) in the level of MT-IIA mRNA by IFN alpha-2a treatment in these cells. Our results suggest that IFN alpha-2a-mediated sensitization of SCC-25 and SCC-4 cell lines to CDDP in vitro may be due to an increase in intracellular platinum accumulation.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Survival; Cisplatin; Glutathione; Glutathione Transferase; Humans; Interferon alpha-2; Interferon-alpha; Metallothionein; Recombinant Proteins; RNA, Messenger; Tumor Cells, Cultured

The expression of metallothionein IIA (MT-IIA) was investigated in A431 human squamous carcinoma cells exposed to hypoxia (pO < or = 0.01% of atmospheric pO2) and subsequent reoxygenation. Northern analysis showed that MT-IIA mRNA levels were significantly increased during 14 h of hypoxia and during reoxygenation. Western blotting confirmed that total MT protein levels were also increased in response to these stresses. Evidence of the transcriptional control of MT-IIA expression in hypoxic and in reoxygenated A431 cells was found using a 0.2-kilobase sequence of the proximal 5'-regulatory region of the MT-IIA gene in a chloramphenicol acetyltransferase reporter gene construct. Thus the proximal promoter of the human MT-IIA gene appears to contain a hypoxic response element(s). These observations indicate that MT-IIA may have an important role in the stress responses of cells in solid tumors.

Topics: Carcinoma, Squamous Cell; Cell Hypoxia; Chloramphenicol O-Acetyltransferase; HSP70 Heat-Shock Proteins; Humans; Metallothionein; Proteins; RNA, Messenger; RNA, Neoplasm; Time Factors; Transfection; Tumor Cells, Cultured; Up-Regulation

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 platinum drug chemosensitivity of five human cervical squamous cell carcinoma cell lines (HX/151, HX/155, HX/156, HX/160 and HX/171) derived from previously untreated patients has been determined. Compared to our data obtained previously using human ovarian carcinoma cell lines, all five lines were relatively resistant to cisplatin, carboplatin, iproplatin and tetraplatin. One of the lines (HX/156) was exceptionally sensitive to the novel platinum (IV) ammine/amine dicarboxylates JM216 [bis-acetatoammine dichloro (cyclohexylamine) platinum (IV)] and JM221 [ammine dibutyrato dichloro (cyclohexylamine) platinum (IV)]. The range in IC50 values across the five lines was approximately 2.5-fold for cisplatin, carboplatin and iproplatin, 13-fold for tetraplatin and JM216, and 25-fold for JM221. No significant correlation (P > 0.05) was observed between platinum drug chemosensitivity and either glutathione levels or cadmium chloride sensitivity, an indicator of metallothionein levels. In addition, there was no significant correlation (P > 0.05) between cisplatin cytotoxicity and intracellular cisplatin accumulation or JM216 cytotoxicity and intracellular JM216 accumulation over the dose range 5-100 microM (2 h exposure). The exceptional sensitivity of HX/156 to JM216 appears, at least partially, to be a result of enhanced accumulation of JM216. An 8.6-fold acquired cisplatin resistant stable variant of HX/155 has been generated in vitro. Intracellular cisplatin accumulation was reduced by 2.4 +/- 0.3-fold (mean +/- s.d.) in HX/155cisR across the dose range 1-100 microM (2 h exposure). Glutathione levels in HX/155cisR were elevated by 1.3-fold in terms of protein content and by 1.6-fold in terms of cell number. HX/155cisR was 1.9-fold resistant to cadmium chloride. Total platinum bound to DNA after cisplatin exposure (10, 25, 50 or 100 microM for 2 h) was 3.6 +/- 0.6-fold (mean +/- s.d.) lower in HX/155cisR. Hence the mechanism of acquired cisplatin resistance in HX/155cisR appears to be multifocal, with reduced intracellular drug accumulation and elevated glutathione and metallothionein levels combining to reduce DNA platination levels. While HX/155cisR was cross-resistant to tetraplatin and carboplatin, novel platinum (II) and (IV) ammine/amine complexes, including JM216 and JM221, partially circumvented resistance (resistance factors of 1.5-2). Non cross-resistance was observed to iproplatin and nine non-platinum anticancer agents. Intracellular tetraplatin

Topics: Antineoplastic Agents; Carboplatin; Carcinoma, Squamous Cell; Cell Division; Cell Survival; Cisplatin; Drug Resistance; Drug Screening Assays, Antitumor; Female; Glutathione; Humans; Metallothionein; Molecular Structure; Organoplatinum Compounds; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Forty-eight human squamous cell lung carcinomas of previously untreated patients were analyzed for resistance to doxorubicin and for the presence of topoisomerase II (Topo II), metallothionein (MT), thymidylate synthase (TS) and catalase (Cat). Significant correlations exist between resistance to doxorubicin measured by the in vitro short-term test and overexpression of MT and TS measured by immunohistochemistry. No significant correlation was found between resistance and expression of Topo II or Cat. No significant interrelationship between smoking habits of patients and expression of Topo II, MT, TS or Cat was found.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Catalase; DNA Topoisomerases, Type II; Doxorubicin; Drug Resistance; Enzymes; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Metallothionein; Middle Aged; Smoking; Thymidylate Synthase

The relationship between intrinsic radiosensitivity and metallothionein (MT) level in several clones isolated from a mouse squamous cell carcinoma was studied. Dose-survival curves for exponentially growing cells assayed by the soft-agar colony method were fitted to the linear-quadratic model by the least-squares method. Intrinsic radiosensitivity was expressed as the mean inactivation dose (D) calculated by the parameters alpha and beta. The D of the clones ranged from 1.6 to 3.9 Gy. A significant negative correlation was obtained between individual D and MT levels determined by the 203Hg-binding assay when expressed as MT content per cell; however, no correlation was obtained when the levels were expressed as MT content per unit cellular volume. The D of the parent cell line MSCC was 2.0 Gy, and that of the most radioresistant clone R1 was 3.9 Gy. Metallothionein content was 315 and 37 Hg-bound pmol/10(6) cells for MSCC and R1 cells, respectively, and thus D was not positively correlated with the level of MT. Metallothionein content of MSCC and R1 cells was elevated to 2304 and 854 Hg-bound pmol/10(6) cells, respectively, by cadmium treatment. Both MSCC and R1 cells with elevated MT content became more resistant to cadmium toxicity. However, the D's of the two cell lines were identical to those of the untreated controls despite the elevated MT content. These results suggest that, at least in this tumor, the endogenous MT level is not a major factor in determining intrinsic radiosensitivity of aerated cells.

Topics: Animals; Cadmium; Carcinoma, Squamous Cell; Cell Survival; Clone Cells; Dose-Response Relationship, Radiation; Metallothionein; Mice; Neoplasm Transplantation; Radiation Tolerance; Stimulation, Chemical

An expression vector was constructed from part of pSV2neo with the 3'-ClaI fragment of the epidermal growth factor (EGF) receptor cDNA inserted in an inverted orientation downstream from the human metallothionein (MT) IIa promoter. The human squamous carcinoma cell line NA, which overproduces EGF receptor, was transfected with this vector and selected for resistance to the neomycin derivative G418. One of the stable transfectants had a 90% reduction in cell-surface EGF receptor in response to ZnSO4. The nascent EGF receptor peptide was also decreased with concurrent induction of MT mRNA. These data suggest that the antisense transcript regulated by the MT promoter inhibits the expression of the endogenous EGF receptor genes. Although no transcripts from the antisense gene were detected, the results indicate that transfection with the antisense vector provides a technique by which to modulate the number of EGF receptors on the cell surface of squamous cell carcinomas.

Topics: Blotting, Northern; Blotting, Southern; Carcinoma, Squamous Cell; Cloning, Molecular; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Genetic Vectors; Humans; Metallothionein; RNA; RNA, Antisense; RNA, Neoplasm; Tumor Cells, Cultured; Zinc

We have developed a human head and neck squamous cell carcinoma cell line (SCC-25/CP) which is relatively stably resistant to cis-diamminedichloroplatinum(II) (CDDP) after repeated exposure to escalating doses of the drug. The studies reported elucidate the mechanism(s) by which the SCC-25/CP cell line is resistant to CDDP. The SCC-25/CP cell line is approximately 30-fold resistant to CDDP, approximately 10-fold resistant to carboplatin, and about 9-fold resistant to iproplatin. Using [195mPt]CDDP, we examined the levels of platinum in whole cells and cellular fractions of both the SCC-25 and SCC-25/CP cells after 1 h exposure to 100 microM drug. The SCC-25 cells took up 30 pmol of platinum/10(6) cells in 1 h; 64% of the drug was in the nucleus and 21% in the cytosol. The SCC-25/CP cells took up 7 pmol of platinum/10(6) cells; of this, 41% was in the nucleus and 33% in the cytosol. The SCC-25 cell nuclei contained 331 pmol of platinum/mg protein and the cytosol 21 pmol of platinum/mg protein, whereas the SCC-25/CP cell nuclei contained 47 pmol of platinum/mg protein and the cytosol 8.1 pmol/mg protein. The release of drug from both cell lines followed a very similar course and was most rapid over the first 6 h. There was no difference in the non-protein sulfhydryl content of the cell lines. The protein sulfhydryl content, as measured by Ellman's procedure, indicated that the SCC-25/CP cell line has approximately a 2-fold increase in protein sulfhydryl content compared to the SCC-25 cell line. The SCC-25/CP cell line is about 2-fold resistant to cadmium chloride at 50% cell kill and about 2.5-fold resistant at 1 log kill compared to the SCC-25 cell line. Glutathione transferase activity in crude cytoplasmic extracts was measured and found to be approximately 2- to 3-fold higher in the CDDP resistant cells. The isoelectric point of the glutathione transferase isozyme was 4.8 in both the sensitive and resistant cell lines, suggesting induction of the predominant isozyme present in the parent cell line. By alkaline elution there was greater cross-link formation by CDDP in the SCC-25 cell line than in the SCC-25/CP cell line at the same drug concentrations. In conclusion, the mechanism of resistance of the SCC-25/CP cell line to CDDP is multifactorial, involving plasma membrane changes, increased cytosolic binding, and decreased DNA cross-linking.

Topics: Biological Transport; Cadmium; Carboplatin; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cisplatin; Drug Resistance; Glutathione Transferase; Humans; Metallothionein; Organoplatinum Compounds; Platinum; Structure-Activity Relationship; Sulfhydryl Compounds