Testis Tumour Cells Research Articles

Strong apoptotic response of testis tumor cells following cisplatin treatment

Most solid metastatic cancers are resistant to chemotherapy. However, metastatic testicular germ cell tumors (TGCT) are cured in over 80% of patients using cisplatin-based combination therapy. Published data suggest that TGCTs are sensitive to cisplatin due to limited DNA repair and presumably also to a propensity to undergo apoptosis. To further investigate this aspect, cisplatin-induced activation of apoptotic pathways was investigated in cisplatin-sensitive testis tumor cells (TTC) and compared to cisplatin-resistant bladder cancer cells. Apoptosis induction was investigated using flow cytometry, caspase activation and PARP-1 cleavage. Immunoblotting and RT-PCR were applied to investigate pro- and anti-apoptotic proteins. Transfections were performed to target p53- and Fas/FasL-mediated apoptotic signaling. Immunoblotting experiments revealed p53 to be induced in TTC, but not bladder cancer cells following cisplatin. Higher levels of pro-apoptotic Bax and Noxa were observed in TTC, anti-apoptotic Bcl-2 was solely expressed in bladder cancer cells. Cisplatin led to translocation of Bax to the mitochondrial membrane in TTC, resulting in cytochrome C release. Cisplatin increased the expression of FasR mRNA and FasL protein in all tumor cell lines. Targeting the apoptotic pathway via siRNA-mediated knockdown of p53 and FAS reduced death receptor-mediated apoptosis and increased cisplatin resistance in TTC, indicating the involvement of FAS-mediated apoptosis in the cisplatin TTC response. In conclusion, both the death receptor and the mitochondrial apoptotic pathway become strongly activated in TTC following cisplatin treatment, explaining, together with attenuated DNA repair, their unique sensitivity toward platinum-based anticancer drugs.

First cytogenomic characterization of murine testis tumor cell line MLTC-1

The cell line MLTC-1 was established in 1982 as a transplantable Leydig cell tumor from a C57BL/6 mouse. The cell line has already been applied in >100 studies: still, the only information about its genetic content is given in the first description: MLTC-1 initially had a polyploid karyotype. Here, a molecular karyotyping and multicolor banding-based molecular cytogenetic study was done to provide the first chromosomal/ (molecular) cytogenetic characterization of MLTC-1. A hexaploid karyotype with 72 to 105 chromosomes was hereby characterized. Besides polyploidy, unbalanced two- and three-way translocations, dicentrics and one neocentric derivative were identified. Also, no Y-chromosome could be detected in this clearly male derived cell line. Overall, a completely unbalanced genome is present in MLTC-1 with ~20 regions being subject to copy number losses or gains. After translating these imbalances into the human genome in a standardized way, a 40% match of imbalances with human Leydig cell tumors was evident; however, about the same rate of concordance was detectable for human spermatocytic seminomas and non-seminomas as well as testicular germ cell tumor. Accordingly, MLTC-1 is better suited as an advanced testicular germ cell tumor model in general, rather than specifically for human Leydig cell tumors.

Cisplatin sensitivity of testis tumour cells is due to deficiency in interstrand-crosslink repair and low ERCC1-XPF expression

BackgroundCisplatin based chemotherapy cures over 80% of metastatic testicular germ cell tumours (TGCT). In contrast, almost all other solid cancers in adults are incurable once they have spread beyond the primary site. Cell lines derived from TGCTs are hypersensitive to cisplatin reflecting the clinical response. Earlier findings suggested that a reduced repair capacity might contribute to the cisplatin hypersensitivity of testis tumour cells (TTC), but the critical DNA damage has not been defined. This study was aimed at investigating the formation and repair of intrastrand and interstrand crosslinks (ICLs) induced by cisplatin in TTC and their contribution to TTC hypersensitivity.ResultsWe observed that repair of intrastrand crosslinks is similar in cisplatin sensitive TTC and resistant bladder cancer cells, whereas repair of ICLs was significantly reduced in TTC. γH2AX formation, which serves as a marker of DNA breaks formed in response to ICLs, persisted in cisplatin-treated TTC and correlated with sustained phosphorylation of Chk2 and enhanced PARP-1 cleavage. Expression of the nucleotide excision repair factor ERCC1-XPF, which is implicated in the processing of ICLs, is reduced in TTC. To analyse the causal role of ERCC1-XPF for ICL repair and cisplatin sensitivity, we over-expressed ERCC1-XPF in TTC by transient transfection. Over-expression increased ICL repair and rendered TTC more resistant to cisplatin, which suggests that ERCC1-XPF is rate-limiting for repair of ICLs resulting in the observed cisplatin hypersensitivity of TTC.ConclusionOur data indicate for the first time that the exceptional sensitivity of TTC and, therefore, very likely the curability of TGCT rests on their limited ICL repair due to low level of expression of ERCC1-XPF.

Metastases of squamous cell carcinoma of the head and neck show increased levels of nucleotide excision repair protein XPF in vivo that correlate with increased chemoresistance ex vivo

Nucleotide excision repair (NER) is a key node of resistance of tumour cells to the anticancer drug cisplatin. Testicular germ cell tumours (TGCT) show exquisite sensitivity towards cisplatin, and this has been connected to low levels of the NER proteins ERCC1 and XPF. Tumours of some patients with advanced head and neck squamous cell carcinoma (HNSCC) regress well under cisplatin chemotherapy but prediction of responsiveness is poor. Little is known about the levels of ERCC1-XPF in HNSCC tissues and cell lines. We investigated mRNA and protein levels of ERCC1 and XPF in 13 HNSCC cell lines and seven testis tumour cell lines and examined the correlation between levels of ERCC1 and XPF and cellular resistance towards cisplatin. No significant difference in mRNA expression of either ERCC1 or XPF in the HNSCC cell lines compared to the testis tumour cell lines was observed. Significantly higher XPF protein levels were found in HNSCC cell lines compared to testis tumour cell lines resulting in cellular cisplatin resistance. The data indicate a contribution of XPF protein for the cisplatin resistance observed in HNSCC cell lines. Subsequently, XPF and ERCC1 protein expression was investigated in cancer tissue of 34 patients. XPF levels were significantly higher in metastases of HNSCC patients than in primary cancer tissue. These findings indicate a contribution of XPF protein for the observed chemoresistance in some HNSCC tissue. XPF protein may be a predictive marker for cisplatin responsiveness of metastases in HNSCC patients.

Characterization of a novel rat gene RTAP2a, screened by cross-reactive SEREX, restrictedly expressed in testis

Genes involved in tumorigenesis may be targets for cancer immunotherapy. For many tumor types, molecular signatures do not yet exist. Methods that identify novel, tissue-specific tumor-related genes therefore have potentially important implications for developing cancer immunotherapy. Here, we used cross-reactive serological analysis of recombinant cDNA expression (CR-SEREX) to identify the novel testicular cancer-related gene, rat testicular antigenic protein 2 (RTAP2). We identified two RTAP2 splice variants (RTAP2a and RTAP2b) encoding 191 and 358 amino acid proteins, respectively. RTAP2a and RTAP2b proteins have identical N-termini but different C-terminal regions arising from alternative splicing of exon 4. Bioinformatics and reverse transcription-polymerase chain reaction (RT-PCR) revealed that the RTAP2a transcript was expressed selectively in adult testis and several tumor cell lines, while RTAP2b was ubiquitous. Our results suggest that RTAP2a may contribute to testicular development and tumorigenesis, and may be a candidate gene for cancer immunotherapy.

Elevation of XPA protein level in testis tumor cells without increasing resistance to cisplatin or UV radiation

Most testicular germ cell tumors are curable using cisplatin-based chemotherapy, and cell lines from these tumors are unusually sensitive to cisplatin and other DNA-damaging agents. It has been suggested that this might be caused by a lower-than normal nucleotide excision repair (NER) activity. Previous studies found that cell lines from testicular germ cell tumors have on average about one-third the level of the NER protein XPA in comparison to cell lines from other tumors. We asked whether over-expression of XPA protein would alleviate the cellular sensitivity and increase the DNA repair capacity of a testis tumor cell line. Increasing XPA levels in 833K cells by 10-fold did not increase resistance to UV irradiation. XPA was localized to the cell nucleus in all cell lines, before and after exposure to UV-radiation. 833K cells were proficient in removing UV radiation-induced photoproducts from the genome and increased XPA did not enhance the rate of removal. Further, over-expressing functional XPA protein did not correlate with increased resistance of 833K testis tumor cells to cisplatin. Thus, although the amount of XPA in this testis tumor cell line is lower than normal, it is sufficient for NER in vivo. The relative sensitivity of testis tumor cells to cisplatin, UV radiation, and other DNA damaging agents is likely related not to NER capacity, but to other factors such as the integrity of the p53 pathway in these cells.

An Aurora Kinase Homologue Is Involved in Regulating Both Mitosis and Cytokinesis in Trypanosoma brucei

The chromosomal passenger protein aurora kinases have been implicated in regulating chromosome segregation and cell division. Three aurora kinase homologues were identified (TbAUK1, -2 and -3) in the Trypanosome Genomic Data Base, and their expressions in the procyclic form of Trypanosoma brucei were knocked down individually by using the RNA interference technique. Only a knockdown of TbAUK1 arrested the cells in G(2)/M phase with each cell showing an extended posterior end, two kinetoplasts, and an enlarged nucleus, apparently the result of an inhibited kinetoplast multiplication and a failed mitosis. There is no mitotic spindle structure in the TbAUK1-depleted cell. The two kinetoplasts moved apart from each other but stopped just before cytokinesis, suggesting that cytokinesis was blocked in its early phase. Overexpression of TbAUK1 in the cells resulted in little change in cell growth. By immunofluorescence, TbAUK1 was primarily localized to the nucleus in interphase and to the mitotic spindle during apparent metaphase and anaphase. Thus, differing from other eukaryotes, TbAUK1 has an apparent triple function in coupling mitosis and kinetoplast replication with cytokinesis in T. brucei. T. brucei polo-like kinase, previously identified as the initiator of cytokinesis without apparent involvement in mitosis in the trypanosome, was either depleted or overexpressed in the TbAUK1-deficient cells. A dominant TbAUK1-depleted phenotype was demonstrated in both cases, suggesting that TbAUK1 plays an essential role in cytokinesis that cannot be affected by changes in the level of T. brucei polo-like kinase. To our knowledge, this is the first time that the function of an aurora B-like kinase is a prerequisite for polo-like kinase action in initiating cytokinesis. TbAUK1 is also the first identified protein that couples both mitosis and kinetoplast replication with cytokinesis in the trypanosome.

Regulation of DNA repair gene expression in human cancer cell lines

Although most advanced cancers are incurable, the majority of testicular germ cell tumors can be cured using cisplatin-based combination chemotherapy. The nucleotide excision repair (NER) pathway removes most DNA adducts produced by cisplatin, and the low levels of NER in testis tumor cells may explain why these cancers are curable. Three NER proteins: ERCC1, XPF, and XPA, are present at low levels in testis tumor cell lines, and addition of these proteins to protein extracts of testis tumor cells increases their in vitro DNA repair capacity to normal levels. The aim of this study was to identify the mechanism responsible for the low levels of these DNA repair proteins. The levels of the mRNA transcripts for ERCC1, XPF, and XPA were measured in a panel of 14 different human cancer cell lines, using real-time PCR. Three ERCC1 splice variants were identified and quantitated. Three alternative transcription start points (TSPs) were identified for ERCC1 but none were testis-specific. The significantly lower levels of ERCC1, XPF, and XPA protein in testis tumor cell lines cannot be explained solely by differences in transcriptional efficiency or mRNA stability. For ERCC1, post-transcriptional control by alternative splicing does not account for the testis-specific low levels of protein expression. Pulse-chase experiments showed that the half-life of ERCC1 protein in a testis tumor cell line was not significantly different to that in a prostate cancer cell line. Taken together, these results suggest that constitutive levels of these DNA repair proteins are controlled at the level of translation.

Apoptosis: a key effector mechanism of lymphocyte action in human nonseminomatous testicular carcinoma?

To correlate the number of tumour-infiltrating T lymphocytes (TILs) with the extent of apoptosis in testicular germ cell tumours, as TILs are considered to be a favourable prognostic factor of human testicular tumours, especially of seminomas, but the mechanism by which TIL contribute to an improved outcome is unclear. Tissue samples from 47 patients with nonseminomatous germ cell tumour (NSGCT) and 15 with seminomatous GCT were investigated immunohistochemically for lymphocyte infiltration and apoptosis. The apoptotic index (AI) was assessed in various categories (DNA condensation and fragmentation) using in-situ end-labelling to identify typical apoptotic DNA strand breaks, and nuclear staining to identify typical apoptotic morphology. In seminomatous GCT there was no correlation between the number of TILs and any AI. In NSGCT there was only a relationship between lymphoid infiltration and those AIs showing morphological criteria of apoptosis in a small subgroup of NSGCT, i.e. metastasized embryonal cell carcinomas. Only 1.2% (AI, chromatin condensation) and 0.8% (AI, fragmentation and condensation) of all tumour cells showed these features of apoptosis. The overall AI in NSGCT was 7.9%. TILs do not seem to induce apoptosis in testicular tumours. Embryonal cell carcinomas might be susceptible to lymphocyte attack, resulting in apoptosis of the tumour cell. The mechanisms of interaction between lymphocytes and testis tumour cells need further investigation.

Reduced levels of XPA, ERCC1 and XPF DNA repair proteins in testis tumor cell lines.

Over 80% of patients with advanced metastatic testis tumors can be cured using cisplatin-based combination chemotherapy. This is unusual as metastatic cancer in adults is usually incurable. Cell lines derived from testis tumors retain sensitivity to cisplatin in vitro. We previously investigated 2 testis tumor cell lines with a low capacity to remove cisplatin-induced DNA damage and found that they had low levels of the DNA nucleotide excision repair proteins XPA, ERCC1 and XPF. To determine whether low levels of XPA, ERCC1 and XPF proteins are characteristic of testis tumor cell lines, we investigated 35 cell lines derived from cancers to determine whether groups of cell lines from diverse tissue origins differ from one another in constitutive levels of these NER proteins. Quantitative immunoblotting was used to compare groups of cell lines representing prostate, bladder, breast, lung, cervical, ovarian and testis cancers. Only the 6 testis tumor cell lines showed significantly lower mean levels of XPA (p = 0.001), XPF (p = 0.001) and ERCC1 (p = 0.004) proteins from the other groups. Our results encourage further investigation of the possibility that low levels of these nucleotide excision repair proteins could be related to the favorable response of testis tumors to cisplatin-based chemotherapy.

Profile of Placental Alkaline Phosphatase Expression in Human Malignancies: Effect of Tumour Cell Activation on Alkaline Phosphatase Expression

Cellular alkaline phosphatases (ALP) are increasingly recognised as important markers for monitoring tumour cell behaviour in human malignancies. Colorimetric, flow-cytometric, and immunocytochemical assays were employed to assess the influence of activation on expression of cellular ALP in human tumour cell lines. The results showed the following: (1) Testis tumour biopsies (16/16) unlike bladder (0/14) and head and neck (0/16) tumours showed positive staining for ALP, particularly the placental type, i.e. PLAP, although this was not always present on all the cells of non-seminoma biopsies. (2) The intensity of ALP expression differed widely in tumour cell lines. Based on biochemical analysis, the profile of ALP fell into two categories: (a) low expressing (MW 70 kD, placental type ALP) like Hep2 and KB lines, and (b) those expressing both low and high molecular (MW 95 kD) bands like testis lines Tera II and Ep2102. In all cases treatment of tumour cell lysates with heat prior to biochemical analysis showed the disappearance of the higher and sharpening of the lower molecular weight ALP band. (3) Exposure of tumour cells to epidermal growth factor (EGF) expressing EGF receptor led to a decreased ALP expression by as much as 54% as assessed by biochemical or flow-cytometric techniques. These data demonstrated that testis tumour tissues and cell lines expressed ALP which were different from others. The data also showed that exposure of tumour cell lines expressing EGFr to EGF resulted in suppression of ALP expression. These observations are consistent with the notion that EGFr and PLAP expression may be taken as a marker of proliferation and differentiation in human malignancies, respectively.

Standardization and Potential Use of HPLC for Detection of Cellular Placental Alkaline Phosphatase Using Established Tumour Cell Lines and Fresh Tumour Biopsies

Placental alkaline phosphatase (PLAP) is one of the cellular phosphatases (ALP) expressed in patients with testis cancers, particularly in seminomas. Using various techniques including Western blot and high-performance liquid chromatography (HPLC) systems and ATC2, a newly developed specific anti-PLAP monoclonal antibody (Mab), the presence of active form of PLAP in lysates prepared from testis tumour fragment and tumour cell lines, was studied. This was carried out following isolation of PLAP from biological samples using CNBr Sepharose-conjugated ATC2 beads. The results showed that: (1) The target for the newly developed Mab ATC2 was PLAP. (2) The ATC2-conjugated bead system was an efficient method for isolating pure PLAP. (3) Diethylamine (DEA), in contrast to urea and glycine, was the most efficient for separation of PLAP from ATC2-conjugated beads, as the isolated molecule did not lose any phosphatase activity and there was very little uncoupling of the ATC2 Mab from the beads. (4) ATC2-conjugated CNBr beads could pick up PLAP from a solution containing standard PLAP and lysates prepared from tumour cell lines or testis tissue fragments positive for the PLAP. (5) HPLC profile of testis tumour lines and testis tumours showed two distinct peaks with ALP activity, one at retention time 7– 8 min (corresponding to 95 kDa molecule) and one at 12–13 min corresponding to 70 kDa molecule). These data demonstrated the potential use of various biochemical methods in combination with HPLC for isolation of the fully functional molecules with ALP activity from different samples including lysates prepared from patients with testis cancer. The nature of ALP activity at 95 kDa is being investigated as no such molecule has been reported previously. These techniques might have an important implication for an early detection of germ cell tumours, particularly in patients with equivocal ultrasound.

Defective repair of cisplatin-induced DNA damage caused by reduced XPA protein in testicular germ cell tumours

Metastatic cancer in adults usually has a fatal outcome. In contrast, advanced testicular germ cell tumours are cured in over 80% of patients using cisplatin-based combination chemotherapy [1]. An understanding of why these cells are sensitive to chemotherapeutic drugs is likely to have implications for the treatment of other types of cancer. Earlier measurements indicate that testis tumour cells are hypersensitive to cisplatin and have a low capacity to remove cisplatin-induced DNA damage from the genome [2,3]. We have investigated the nucleotide excision repair (NER) capacity of extracts from the well-defined 833K and GCT27 human testis tumour cell lines. Both had a reduced ability to carry out the incision steps of NER in comparison with extracts from known repair-proficient cells. Immunoblotting revealed that the testis tumour cells had normal amounts of most NER proteins, but low levels of the xeroderma pigmentosum group A protein (XPA) and the ERCC1–XPF endonuclease complex. Addition of XPA specifically conferred full NER capacity on the testis tumour extracts. These results show that a low XPA level in the testis tumour cell lines is sufficient to explain their poor ability to remove cisplatin adducts from DNA and might be a major reason for the high cisplatin sensitivity of testis tumours. Targeted inhibition of XPA could sensitise other types of cells and tumours to cisplatin and broaden the usefulness of this chemotherapeutic agent.

Complementation analysis of testis tumor cells

Testis tumors are cured using cisplatin-based chemotherapy in over 80% of patients, and sensitivity to cisplatin is retained by testis tumor cells in vitro. The aim of this study was to use complementation analysis to determine how many genes control sensitivity to cisplatin in testis tumor cells. Four testis tumor cell lines were transfected with pSV2NEO and pBABE plasmids, conferring G418 and puromycin resistance, respectively. Self-crosses were generated to control for gene dosage, and the parentage of the hybrids was confirmed by PCR amplification of VNTR regions. Karyotyping confirmed that all the hybrids retained at least 88% of the combined number of chromosomes of the two parental cell lines. Cisplatin sensitivity was measured by clonogenic assay and complementation was not observed. This finding provides evidence that there is a single common mechanism controlling cisplatin sensitivity in testis tumor cells.

DNA Repair in Cisplatin-Sensitive and Resistant Human Cell Lines Measured in Specific Genes by Quantitative Polymerase Chain Reaction

More than 80% of patients with testicular germ cell tumours (TGCT) are cured using cisplatin-based combination chemotherapy, and resistance to cisplatin is the final barrier to the cure of nearly all patients with this disease. In this study, we used quantitative polymerase chain reaction (Q-PCR) to investigate the role of DNA repair in cisplatin resistance in two genes, one transcribed and one not transcribed. Three pairs of cisplatin-sensitive and resistant cell lines were used, two derived from TGCT and one from a bladder cancer. In these pairs of sublines, we observed no major differences between the repair of cisplatin-induced damage in the transcribed and nontranscribed genes, nor did there appear to be any relationship between DNA repair capacity and the development of cisplatin resistance. Despite the strong indication that the sensitivity of testis tumour cells to cisplatin is related to their reduced ability to repair cisplatin-damaged DNA, these cells apparently do not become resistant to cisplatin by acquiring DNA repair capacity.

Thermotolerance and sensitivity of human cancer cells to cisplatin and doxorubicin

Testis tumour cells are more sensitive than most other types of cancer cell to heat, radiation and chemotherapeutic drugs, both in the clinic and in vitro. Since heat shock proteins (HSP) can protect cells from the cytotoxic effects of stress, we studied their role in the sensitivity of testis tumour cells to the frequently used cancer chemotherapeutic drugs, cisplatin and doxorubicin. Clonogenic assays of 3 testis tumour cell lines (833K, GCT27, GH) and 3 bladder cancer cell lines (RT112, HT1376, MGH-U1) following a 1 h exposure to the two drugs confirmed that the testis tumour cell lines were inherently more sensitive. Having shown previously that heat shock proteins were upregulated in both cell types following a heat shock, in this study no induction of HSP was seen following treatment for 1 h with cisplatin or doxorubicin (at concentrations reducing colony forming ability by 50%) in either cell type. In contrast, chronic exposure to cisplatin (at concentrations on the threshold of cytotoxicity), but not doxorubicin, resulted in upregulation of HSP 27, but not HSP73/72, in both cell types. Testis and bladder cancer cells with heat-induced increases in HSP were thermotolerant, and this was associated with increased resistance to doxorubicin, but not cisplatin.

Sensitivity of testis tumour cells to chemotherapeutic drugs: Role of detoxifying pathways

In contrast to most other types of cancer, metastatic testicular germ cell tumours (TGCT) are cured in most patients using cisplatin-based combination chemotherapy. The biochemical mechanisms underlying this sensitivity have not been denned. Drug detoxification can modulate response to chemotherapy in vivo and in vitro, and therefore we measured levels of glutathione (GSH), glutathione- S-transferase (GST) and both constitutive and cisplatin- and dexamethasone-induced levels of metallo- thionein (MT) in five human testis tumour cell lines. The levels were compared with those in five human bladder cancer cell lines and two cell lines with cisplatin resistance acquired in vitro. GSH levels were relatively low in the testis tumour cell lines, as might be expected in drug-sensitive cells, and there was a 77-fold increase in GSH levels in the cisplatin-resistant testis tumour cell line. GST levels were similar in the two cell types, while metallothionein levels were relatively high in the testis tumour cell lines. These data indicate that GSH may contribute to the sensitivity of TGCT to chemotherapy, and that GSH expression may be involved in the acquisition of cisplatin resistance in these tumours.

Genetic basis of drug sensitivity in human testis tumour cells.

Testicular germ cell tumours (TGCT) are cured in over 80% of patients by using combination chemotherapy. However, the mechanism regulating this sensitivity has not been defined. Because cells derived from patients with DNA repair syndromes are similar to TCGT in their sensitivity to certain DNA-damaging agents and some of the genes involved have been cloned by functional complementation, the purpose of our study was to determine whether drug sensitivity in TGCT also has a genetic basis. Three testis tumour cell lines (cisplatin-sensitive) and 3 bladder cancer cell lines (cisplatin-resistant) were fused with a cisplatin-sensitive cell line (D98orC1). The authenticity of the hybrids was confirmed by karyotyping and PCR analysis of locus-specific sites, and sensitivities to cisplatin were measured by colony forming assays. The hybrids between sensitive cell lines were more resistant to cisplatin than the parental cells, indicating that functional complementation had occurred. The hybrids between the cisplatin-resistant and sensitive cells were intermediate in their cisplatin sensitivity, indicating that resistance is incompletely dominant. We conclude that cisplatin sensitivity has a genetic basis in TGCT and that resistance to cisplatin can be conferred by somatic cell fusion. Our data indicate that gene(s) controlling sensitivity to chemotherapy in TGCT might be identified by expression cloning.

Genetic basis of drug sensitivity in human testis tumour cells

Genetic basis of drug sensitivity in human testis tumour cells

Heat shock protein expression in testis and bladder cancer cell lines exhibiting differential sensitivity to heat

Testis cancer cells are more sensitive than bladder and most other cancer cells to chemotherapeutic drugs both in the clinic and in vitro. In this study we show that they are also more sensitive than bladder cancer cells to heat. Since heat and drug sensitivity may be related to the ability of a cell to mount a stress response, constitutive and induced levels of heat shock proteins (HSPs) in three testis and three bladder human cancer cell lines were measured using Western blotting and scanning densitometry. No correlation between constitutive levels of HSP 90 or HSP 73/72 and cellular heat sensitivity was found. However, HSP 27 levels were much lower in the testis tumour cells, suggesting that low HSP 27 expression might contribute to heat sensitivity. Protein synthesis studies using [35S]methionine indicated that, for the same heat shocks, the kinetics of synthesis and decay of HSP 90 and HSP 73/72 in 833K (the most heat sensitive testis cells) was similar to or greater than that in HT1376 (the most heat-resistant bladder cells). Both 833K and HT1376 developed thermotolerance, and this followed an increase in synthesis of HSPs. These results indicate that, although there are differences in the constitutive levels of HSPs between testis and bladder cancer cells, both cell types are capable of mounting an induced heat shock response and can develop a similar degree of thermotolerance.