Triazene compounds in the treatment of acute myeloid leukemia: a short review and a case report.

Acute myeloid leukemia (AML) is a highly lethal disease, especially in old patients. Chemoresistance and the absence of host immune responses against autochthonous malignancy play a major role in the poor prognosis of AML. The triazene compounds Dacarbazine and Temozolomide are monofunctional alkylators that donate methyl groups to many sites in DNA, including the O(6)-position of guanine producing O(6)-methylguanine (O(6)-MeG). If not repaired, O(6)-MeG frequently mispairs with thymine during DNA duplication. O(6)-MeG:T mismatches can be recognized by the mismatch repair (MMR) system which activates a cascade of molecular events leading to cell cycle arrest and cell death. If MMR is defective, cells continue to divide and GC → AT transition mutations occur. In preclinical models, such mutations can lead to the appearance of abnormal proteins containing non-self peptides ("chemical xenogenization" CX) that can be recognized by host cell-mediated immunity. Repair of O(6)-MeG is achieved by the DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT), which removes the methyl adduct in an autoinactivating stoichiometric reaction. High MGMT levels attenuate the pharmacodynamic effects of triazenes. In the last few years, triazenes, alone or with MGMT inhibitors, have been tested in AML. In view of their potential activity as CX inducers, triazenes could offer the additional advantage of host anti-leukemia immune responses. The present paper describes several studies of leukemia treatment with triazenes and a case of acute refractory leukemia with massive skin infiltration by malignant cells. Treatment with Temozolomide and Lomeguatrib, a potent MGMT inhibitor, produced a huge, although transient, blastolysis and complete disappearance of all skin lesions.

Expression of the soluble vascular endothelial growth factor receptor-1 in cutaneous melanoma: role in tumour progression.

BACKGROUND: Vascular endothelial growth factor (VEGF)-A, placenta growth factor (PlGF) and their corresponding membrane receptors are involved in autocrine and paracrine regulation of melanoma growth and metastasis. Besides the membrane receptors, a soluble form of the VEGF receptor (VEGFR)-1 (sVEGFR-1) has been identified, that behaves both as a decoy receptor, sequestering VEGF-A and PlGF, and as an extracellular matrix (ECM) molecule, promoting endothelial cell adhesion and migration through the interaction with α5ß1 integrin. OBJECTIVES: To analyse whether sVEGFR-1 plays a role during melanoma progression. METHODS: sVEGFR-1 expression was evaluated in a panel of 36 melanoma cell lines and 11 primary human melanocyte cultures by quantitative real-time polymerase chain reaction analysis and in specimens of primary or metastatic melanoma lesions from 23 patients by immunohistochemical analysis. RESULTS: sVEGFR-1 expression was highly upregulated in melanoma cell lines with respect to human melanocytes. Interestingly, cell lines obtained from cutaneous metastases showed a significant reduction of sVEGFR-1 expression, as compared with cell lines derived from primary tumours. These results were confirmed by immunohistochemical analysis of sections from primary skin melanomas and the corresponding cutaneous metastases, suggesting that modulation of sVEGFR-1 expression influences ECM invasion by melanoma cells and metastasis localization. Moreover, we provide evidence that adhesion of melanoma cells to sVEGFR-1 is favoured by the activation of a VEGF-A/VEGFR-2 autocrine loop. CONCLUSIONS: Our data strongly suggest that sVEGFR-1 plays a role in melanoma progression and that low sVEGFR-1/VEGF-A and sVEGFR-1/transmembrane VEGFR-1 ratios might predict a poor outcome in patients with melanoma.

Cells deficient in the base excision repair protein, DNA polymerase beta, are hypersensitive to oxaliplatin chemotherapy.

A significant proportion of human cancers overexpress DNA polymerase beta (Pol beta), the major DNA polymerase involved in base excision repair. The underlying mechanism and biological consequences of overexpression of this protein are unknown. We examined whether Pol beta, expressed at levels found in tumor cells, is involved in the repair of DNA damage induced by oxaliplatin treatment and whether the expression status of this protein alters the sensitivity of cells to oxaliplatin. DNA damage induced by oxaliplatin treatment of HCT116 and HT29 colon cancer cells was observed to be associated with the stabilization of Pol beta protein on chromatin. In comparison with HCT116 colon cancer cells, isogenic oxaliplatin-resistant (HCT-OR) cells were found to have higher constitutive levels of Pol beta protein, faster in vitro repair of a DNA substrate containing a single nucleotide gap and faster repair of 1,2-GG oxaliplatin adduct levels in cells. In HCT-OR cells, small interfering RNA knockdown of Pol beta delayed the repair of oxaliplatin-induced DNA damage. In a different model system, Pol beta-deficient fibroblasts were less able to repair 1,2-GG oxaliplatin adducts and were hypersensitive to oxaliplatin treatment compared with isogenic Pol beta-expressing cells. Consistent with previous studies, Pol beta-deficient mouse fibroblasts were not hypersensitive to cisplatin treatment. These data provide the first link between oxaliplatin sensitivity and DNA repair involving Pol beta. They demonstrate that Pol beta modulates the sensitivity of cells to oxaliplatin treatment.

Molecular alterations at chromosome 9p21 in melanocytic naevi and melanoma.

BACKGROUND: The chromosome 9p21 and its CDKN locus, with the p16 tumour suppressor gene (CDKN2A), are recognized as the genomic regions involved in the pathogenesis of melanoma. OBJECTIVES: To elucidate further the role of such regions during the different phases of melanocytic tumorigenesis. METHODS: Tissue sections from naevi, primary and metastatic melanomas were investigated by fluorescence in situ hybridization for allelic loss at the 9p21 chromosome and by immunochemistry for p16CDKN2A expression. RESULTS: Dysplastic naevi and primary or secondary melanomas were found to carry hemizygous deletions within the entire 9p21 region at similar frequencies (varying from 55% to 62%). Allelic deletion spanning the CDKN locus was observed at significantly increased rates moving from early (7%) to advanced (28%) primary melanomas and to secondary melanoma lesions (37%) (P=0.018). Also, inactivation of the p16 gene (CDKN2A) was absent in naevi and present at steadily increasing rates moving from primary melanomas (7% early lesions to 17% advanced lesions) to melanoma metastases (62%) (P=0.004). CONCLUSIONS: Our findings indicate that, in a model of sequential accumulation of genetic alterations, 9p21 deletions may play a role in melanocytic transformation and tumour initiation whereas rearrangements at the CDKN locus, and p16 gene (CDKN2A) inactivation may contribute to tumour progression.

O6-(4-bromothenyl)guanine (PaTrin-2), a novel inhibitor of O6-alkylguanine DNA alkyl-transferase, increases the inhibitory activity of temozolomide against human acute leukaemia cells in vitro.

Anti-tumour activity of triazene compounds of clinical interest [i.e. dacarbazine and temozolomide (TMZ)] relies mainly on the generation of methyl adducts to purine bases of DNA. Two DNA repair enzyme systems, i.e. the O6-guanine-alkyl-transferase (MGMT) and mismatch repair (MMR), play a predominant role in conditioning the cytotoxic effects of triazenes. In particular, high levels of MGMT associated with target cells are responsible of resistance to triazenes. On the contrary, the presence of MMR is required for the cytotoxic effects of these compounds. Previous studies performed by our group and a more recent clinical investigation reported by Karen Seiter, pointed out that triazene compounds could play an important role in the treatment of refractory acute leukaemia. Leukaemia blasts, especially of lymphoblastic leukaemia, show frequently high levels of MGMT activity. Therefore, it reasonable to hypothesize that combined treatment of leukaemia patients with triazene compounds along with MGMT inhibitors could lead to a better control of the disease. PaTrin-2 (O6-(4-bromothenyl)guanine, PAT) is a potent and scarcely toxic MGMT inhibitor recently introduced in clinical trials. This drug is used in combination with triazene compounds in order to augment their anti-tumour efficacy against neoplastic cells endowed with high MGMT activity. The present report describes, for the first time, pre-clinical in vitro studies on the cytotoxic activity of combined treatment with PAT+TMZ against long-term cultured leukaemia cells and primary leukaemia blasts obtained from patients with acute lymphoblastic leukaemia or acute myeloblastic leukaemia. The results point out that, both in long-term cultured leukaemia cell lines and in primary blast samples, PAT could improve dramatically the sensitivity of malignant cells to the cytotoxic effects of TMZ. This sensitizing effect is detectable when leukaemia cells show resistance mechanisms based on a MGMT-proficient phenotype. On the contrary, when resistance to TMZ is dependent on MMR deficiency, no influence of PAT can be detected in various experimental conditions. In conclusion, these results appear to provide disease-oriented rational basis to design novel clinical protocols for the treatment of acute leukaemia with combined administration of PAT and triazene compounds.

Preclinical studies on detection of circulating melanoma cells in patients: telomerase as a recognition marker of malignancy.

Preclinical studies based on a "simulation design", were performed with cultured melanoma cells prelabeled with 51Cr, added to normal blood and subjected to separation and recognition steps. Mononuclear cells (MNC) were isolated on ficollhypaque gradient, and melanoma cells were separated from lymphocytes using anti-CD45 immunomagnetic beads. Malignant cells were then recognized by measuring telomerase activity (TRAP and TRAP-ELISA assays). It was found that: (a)recovery of prelabeled cells present in MNC did not exceed 75%; (b) further recovery of prelabeled cells after separation from lymphocytes did not exceed 68%. Therefore, the overall recovery of prelabeled cells did not exceed 48%; (c) the entire procedure was able to reliably detect as few as 30 malignant cells added to normal blood, providing a telomerase signal significantly higher than that found in absence of melanoma cells. These results furnish the technical bases for developing a tumor detection assay in the blood of melanoma patients.

Adjuvant therapy of melanoma patients (stage II, III): a pilot immuno-toxicological study.

A pilot study was conducted to assess the tolerability and the effect on host immunity of a post-surgery adjuvant treatment of melanoma patients with an anti-angiogenic agent, Tamoxifen (TAM, 20 mg/die p.o., daily), combined with immunomodulating cytokines, i.e. recombinant interleukin-2 (IL-2, 4 MUI/m2 s.c., day 8,10,12) and alpha-2b-interferon (IFN, 3 MUI/m2 i.m., day 15,17,19), starting a new cycle on day 21, for a total of 12 cycles. Fifty patients (pts) entered into the study, 27 males and 23 females with a median age of 55 years (range 25-75), performance status (ECOG) 0 with melanoma stage IIA (12 patients), stage IIB (28 patients), stage III (10 patients). Preliminary in vitro studies showed that TAM does not interfere with up-regulation of natural immunity induced by IFN, IL-2, or IFN + IL-2 in normal peripheral blood mononuclear cells (MNC). The clinical study indicates that the protocol was well tolerated. Increase of NK and LAK activity of patient MNC was observed on day 15. The mean disease-free interval was 10 months and 40 pts were alive at 5 years of follow-up. Further investigations should be performed to test effectiveness of this protocol in a randomized study.

DNA repair enzymes and cytotoxic effects of temozolomide: comparative studies between tumor cells and normal cells of the immune system.

O6-alkylguanine-DNA alkyltransferase (OGAT) and the mismatch repair system (MRS) play a crucial role in the susceptibility of tumor cells to the cytotoxic effects of agents that generate O6-methylguanine in DNA, including the triazene compound temozolomide (TMZ). Studies performed with peripheral blood mononuclear cells (MNC) showed that TMZ was scarcely active on lymphocyte functions not dependent on cell proliferation (e.g. NK activity and cytokine-mediated induction of CD1b molecule in adherent MNC). In contrast, TMZ depressed proliferation and lymphokine activated killer (LAK) cell generation in response to IL-2. In this case, a reasonably good inverse relationship was found between OGAT levels of MNC and their susceptibility to TMZ. This study also analyzed the ratio of the toxic effect of TMZ on MNC and on tumor cells (i.e. "Tumor-Immune Function Toxicity Index", TIFTI). A particularly favorable TIFTI can be obtained when OGAT levels are extremely high in MNC and markedly low in tumor cells. This holds true for MRS-proficient neoplastic cells, but not for MRS-deficient tumors. In conclusion, strategies aimed at modulating OGAT and MRS may improve the clinical response to TMZ. However, the use of OGAT inhibitors to potentiate the antitumor activity of TMZ might result in a concomitant increase of the immunosuppressive effects of the drug, thus reducing the relative TIFTI.

Phenotypic analysis of hMSH2 mutations in mouse cells carrying human chromosomes.

Conversion of diploidy to haploidy is a method that allows the generation of stable murine/human hybrid cell lines carrying selected human chromosomes in only a single copy. In this setting, it is possible to detect genetic mutations with greater sensitivity and reliability than in diploid cells. Using this method, we were able to identify mutations in the human mismatch repair (MMR) gene hMSH2 in hereditary nonpolyposis colon cancer families, which have escaped detection by the conventional methods. In this report, we show that such hybrid cell lines can also be a valuable tool in the study of the mutated MMR proteins, in particular the variants found in hereditary nonpolyposis colon cancer families that carry missense mutations and where it is unclear whether they predispose to colon cancer. This analysis is made possible by the fact that the human hMSH2 protein is able to complement the MMR defect in the host murine cell line.

hMSH3 overexpression and cellular response to cytotoxic anticancer agents.

Mutations or transcriptional silencing of mismatch repair genes have been linked with tumour cell resistance to O(6)-guanine methylating agents, 6-thioguanine, cisplatin, doxorubicin and etoposide. Recently, it has been demonstrated that overexpression of the MSH3 protein is associated with depletion of the mismatch binding factor MutSalpha, and then with a marked reduction in the efficiency of base/base mismatch repair. In the present study we evaluated sensitivity of the HL-60 cell line and its methotrexate-resistant subline HL-60R, which overexpresses the hMSH3 gene, to a panel of chemotherapeutic agents. Cell growth inhibition induced by temozolomide, 6-thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine was significantly lower in the hMSH3-overexpressing HL-60R cell line as compared with the HL-60 parental line. Moreover, HL-60R cells were more resistant than HL-60 cells to chromosome aberrations induced by either N-methyl-N'-nitro-N-nitrosoguanidine or temozolomide, and to apoptosis triggered by the latter drug. Both cell lines were equally susceptible to growth inhibition induced by cisplatin, etoposide or doxorubicin. In addition, HL-60 and HL-60R cells showed comparable sensitivity to the clastogenic and apoptotic effects of cisplatin and etoposide. These results further confirm that loss of base/base mismatch repair is the most important molecular mechanism involved in cell resistance to O(6)-guanine methylating agents and 6-thioguanine. However, the status of the mismatch repair system could still influence tumour cell sensitivity to cisplatin, etoposide and doxorubicin, depending on the specific component of the system that is lost, and on the genetic background of the cell.

Tolerance of human MSH2+/- lymphoblastoid cells to the methylating agent temozolomide.

Members of hereditary nonpolyposis colon cancer (HNPCC) families harboring heterozygous germline mutations in the DNA mismatch repair genes hMSH2 or hMLH1 present with tumors generally two to three decades earlier than individuals with nonfamilial sporadic colon cancer. We searched for phenotypic features that might predispose heterozygous cells from HNPCC kindreds to malignant transformation. hMSH2(+/-) lymphoblastoid cell lines were found to be on average about 4-fold more tolerant than wild-type cells to killing by the methylating agent temozolomide, a phenotype that is invariably linked with impairment of the mismatch repair system. This finding was associated with an average 2-fold decrease of the steady-state level of hMSH2 protein in hMSH2(+/-) cell lines. In contrast, hMLH1(+/-) heterozygous cells were indistinguishable from normal controls in these assays. Thus, despite the fact that HNPCC families harboring mutations in hMSH2 or hMLH1 cannot be distinguished clinically, the early stages of the carcinogenic process in hMSH2 and hMLH1 mutation carriers may be different. Should hMSH2(+/-) colonocytes and lymphoblasts harbor a similar phenotype, the increased tolerance of the former to DNA-damaging agents present in the human colon may play a key role in the initiation of the carcinogenic process.

Human melanoma cells secrete and respond to placenta growth factor and vascular endothelial growth factor.

The vascular endothelial growth factor is produced by a large variety of human tumors, including melanoma, in which it appears to play an important role in the process of tumor-induced angiogenesis. Little information is available on the role of placenta growth factor, a member of the vascular endothelial growth factor family of cytokines, in tumor angiogenesis, even though placenta growth factor/vascular endothelial growth factor heterodimers have been recently isolated from tumor cells. To investigate the role of placenta growth factor and vascular endothelial growth factor homodimers and heterodimers in melanoma angiogenesis and growth, 19 human melanoma cell lines derived from primary or metastatic tumors were characterized for the expression of these cytokines and their receptors. Release of placenta growth factor and vascular endothelial growth factor polypeptides into the supernatant of human melanoma cells was demonstrated. Reverse transcriptase polymerase chain reaction analysis showed the presence of mRNAs encoding at least three different vascular endothelial growth factor isoforms (VEGF(121), VEGF(165), and VEGF(189)) and transcripts for two placenta growth factor isoforms (PlGF-1 and PlGF-2) in human melanoma cells. In addition, placenta growth factor expression in human melanoma in vivo was detected by immunohistochemical staining of tumor specimens. Both primary and metastatic melanoma cells were found to express the mRNAs encoding for vascular endothelial growth factor and placenta growth factor receptors (KDR, Flt-1, neuropilin-1, and neuropilin-2), and exposure of melanoma cells to these cytokines resulted in a specific proliferative response, supporting the hypothesis of a role of these angiogenic factors in melanoma growth. J Invest Dermatol 115:1000-1007 2000

In vitro effect of hyperthermia on natural cell-mediated cytotoxicity.

It is well known that hyperthermia (HY), which is used for the treatment of cancer, depresses natural cell-mediated immunity in vitro. Experiments were performed to confirm the inhibitory effect of HY (42 degrees C for 1 hour) on natural killer (NK) activity and to evaluate the influence of HY on the generation and cytotoxic activity of interleukin-2 (IL-2)-activated NK cells. Additional experiments were also carried out to evaluate the effect of a simultaneous exposure of effector and target cells to HY. The results showed that HY profoundly reduced the lytic activity of NK cells and demonstrated that this inhibition was transient and not due to an apoptosis-induced reduction of the number of effector cells. Moreover, the exposure of mononuclear cells to HY before IL-2 stimulation did not affect the generation of IL-2-activated NK cells, whereas, the hyperthermic treatment of IL-2-activated NK cells produced a marked reduction of their cytotoxic activity. The results also showed that the simultaneous exposure of effector and target cells to HY, during the cytotoxicity assay, produced a marked reduction of lytic activity of NK and IL-2-activated NK cells, and that this impairment was specific for effector cells. In this context, heat-exposure of target cells alone, did not substantially modify their susceptibility to lysis induced by either NK or IL-2-activated NK cells. These results add further evidence of HY-induced inhibition of natural cell-mediated immunity, and suggest that, in the course of therapeutic HY, immune response could be significantly altered.

Attenuation of O(6)-methylguanine-DNA methyltransferase activity and mRNA levels by cisplatin and temozolomide in jurkat cells.

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is important in cellular resistance to certain alkylating antitumor agents such as the methylating drug temozolomide (TMZ). To provide a more rational basis for clinical combinations with another commonly used drug, cisplatin, we assessed the modulation of MGMT protein and mRNA levels in the human leukemic cell line Jurkat after treatment with these agents. Cisplatin decreased MGMT activity in a time- and dose-dependent manner, with maximal suppression (50%) observed 24 h after treatment with 25 microM cisplatin. This was probably the result of decreased transcription of the MGMT gene, because there was an earlier nadir of MGMT mRNA levels after cisplatin treatment and neither cisplatin nor DNA reacted with cisplatin in vitro was able to inhibit MGMT activity in an in vitro assay. TMZ alone depleted MGMT activity in a time- and dose-dependent manner with almost complete loss of activity occurring immediately after treatment with 500 microM TMZ. Combinations of cisplatin (12.5 microM) and TMZ (250 microM) caused substantial and prolonged MGMT depletion with recovery to only 30% of pretreatment levels by 48 h. These results suggest that the clinical efficacy of TMZ and cisplatin may be improved by appropriate schedules of combinations of these agents.

Downregulation of 14-3-3sigma prevents clonal evolution and leads to immortalization of primary human keratinocytes.

In human epidermal keratinocytes, replicative senescence, is determined by a progressive decline of clonogenic and dividing cells. Its timing is controlled by clonal evolution, that is, by the continuous transition from stem cells to transient amplifying cells. We now report that downregulation of 14-3-3sigma, which is specifically expressed in human stratified epithelia, prevents keratinocyte clonal evolution, thereby forcing keratinocytes into the stem cell compartment. This allows primary human keratinocytes to readily escape replicative senescence. 14-3-3sigma-dependent bypass of senescence is accompanied by maintenance of telomerase activity and by downregulation of the p16(INK4a) tumor suppressor gene, hallmarks of keratinocyte immortalization. Taken together, these data therefore suggest that inhibition of a single endogenous gene product fosters immortalization of primary human epithelial cells without the need of exogenous oncogenes and/or oncoviruses.

O(6)-benzylguanine enhances the in vitro immunotoxic activity of temozolomide on natural or antigen-dependent immunity.

Temozolomide (TMZ) is a new cytotoxic triazene compound of clinical interest that is able to generate methyl adducts at the O(6)-guanine of DNA, which can be repaired by O(6)-alkylguanine-DNA alkyltransferase (OGAT). It was previously found that triazene compounds are highly immunosuppressive in mice. In the present study, we investigate whether TMZ could affect immune functions of human competent cells and whether methylation of O(6)-guanine could be involved in the immunosuppressive activity of the drug. Mononuclear cells (MNCs) obtained from peripheral blood of healthy donors were tested for OGAT activity and treated with TMZ alone or combined with the OGAT inhibitor O(6)-benzylguanine. Control or drug-treated MNCs were then assayed for natural killer activity and for the ability to proliferate and to generate cytotoxic effector cells in response to interleukin-2 or allogeneic MT-2 tumor cells. The results show that TMZ inhibited both proliferation and induction of lytic activity in response to interleukin-2 or allogeneic MT-2 cells. Moreover, an inverse correlation was found between the OGAT activity of MNCs and their sensitivity to TMZ. The involvement of O(6)-guanine methylation in the immunosuppressive effects of TMZ was further confirmed by the finding that O(6)-benzylguanine increased the activity of the drug. On the other hand, the natural killer activity of MNCs was only moderately affected by TMZ, and no relationship was observed between OGAT levels and sensitivity to the drug. These data suggest that in patients with tumors who are undergoing TMZ treatment, the drug may impair immune responses involving cell proliferation, depending on OGAT levels of MNCs, and that O(6)-benzylguanine may potentiate this activity.

In vitro infection of CD4+ T lymphocytes with HTLV-I generates immortalized cell lines coexpressing lymphoid and myeloid cell markers.

The human T cell leukemia/lymphoma virus (HTLV-I) is the etiologic agent of adult T cell leukemia (ATL). CD4+ lymphocytes are the preferential targets of infection, even though other cell types can be infected in vitro by the virus. Although ATL cells show CD3 and CD4 surface markers, some ATL-derived cell lines were reported to express also myeloid antigens. In order to analyze possible phenotypic changes induced by HTLV-I after infection of human lymphocytes, CD4+ cells were isolated from peripheral blood of three healthy donors, by separation through immunomagnetic beads. CD4+ lymphocytes were then infected by coculture with irradiated HTLV-I producing MT-2 cells. The phenotypic profile of infected cells was studied by flow cytometric analysis using monoclonal antibodies against lymphoid (CD3, CD4, TCR alpha/beta) and myelomonocitic markers (CD13, CD14, CD15, CD33, CD34). The results show that HTLV-I immortalized cell lines coexpressed CD13, CD33 and lymphoid markers. No expression of CD14, CD15 and CD34 was observed. These data suggest that the presence of both myeloid and lymphoid phenotype in HTLV-I infected T cells is the results of an induction rather than a selection mechanism.

Progressive increase in telomerase activity from benign melanocytic conditions to malignant melanoma.

The expression of telomerase activity and the in situ localization of the human telomerase RNA component (hTR) in melanocytic skin lesions was evaluated in specimens from sixty-three patients. Specimens of melanocytic nevi, primary melanomas and subcutaneous metastases of melanoma were obtained from fifty-eight patients, whereas metastasized lymph nodes were obtained from five patients. Telomerase activity was determined in these specimens by using a Polymerase Chain Reaction-based assay (TRAP). High relative mean telomerase activity levels were detected in metastatic melanoma (subcutaneous metastases = 54.5, lymph node metastases = 56.5). Much lower levels were detected in primary melanomas, which increased with advancing levels of tumor cell penetration (Clark II = 0.02, Clark III = 1.1, and Clark IV = 1.9). Twenty-six formalin-fixed, paraffin-embedded melanocytic lesions were sectioned and analyzed for telomerase RNA with a radioactive in situ hybridization assay. In situ hybridization studies with a probe to the template RNA component of telomerase confirmed that expression was almost exclusively confined to tumor cells and not infiltrating lymphocytes. These results indicate that levels of telomerase activity and telomerase RNA in melanocytic lesions correlate well with clinical stage and could potentially assist in the diagnosis of borderline lesions.

Sensitivity of human melanoma cells to oestrogens, tamoxifen and quercetin: is there any relationship with type I and II oestrogen binding site expression?

We investigated the effect of oestrogens, anti-oestrogens and flavonoids on the growth of a human melanoma cell line (SK-Mel-28) and, at the same time, the presence of both type I oestrogen receptors (ERs) and type II oestrogen binding sites (type II EBS) to gain a fuller picture of the relationship between melanoma cell proliferation and receptor status. 17beta-Oestradiol (E2) and the flavonoid quercetin (Q) produced a marked inhibition of proliferation, but only at the highest dose used (10(-5) M) and only when added daily to the medium. Diethylstilboestrol (DES) (10(-5) M) was effective in inhibiting cell growth when the medium was renewed every 3 days and produced a more pronounced reduction when added daily to the medium. Tamoxifen (TAM) inhibited cell proliferation at a dose starting from 10(-7) M when the medium was renewed every 3 days. When added daily to the medium, it did not induce a greater inhibitory effect and it was cytotoxic at 5 x 10(-6) M and 10(-5) M. The antiproliferative effect of E2, DES and Q did not seem to be dependent on their interaction with ERs, which were minimally detected in SK-Mel-28 in both immunocytochemical and biochemical assays. Our model revealed, through a biochemical assay, a large number of type II EBSs which could be involved in the anti-oestrogen action, but this does not exclude the involvement of other mechanisms. Finally, TAM (10(-5) M) appeared to reduce the activity of the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase, an effect that could be interesting from the point of view of the therapeutic efficacy of alkylating agents.

Mutation of the mismatch repair gene hMSH2 and hMSH6 in a human T-cell leukemia line tolerant to methylating agents.

Cell killing by monofunctional methylating agents is due mainly to the formation of adducts at the O6 position of guanine. These methyl adducts are removed from DNA by the O6-alkylguanine DNA alkyltransferase (OGAT). The mechanism by which O6-methylguanine (O6meG) induces cell death in OGAT-deficient cells requires a functional mismatch repair system (MRS). We have previously reported that depletion of OGAT activity in the human T-cell leukemic urkat line does not sensitize these cells to the cytotoxic and apoptotic effects of the methylating triazene temozolomide (Tentori et al., 1995). We therefore decided to establish whether the tolerance of Jurkat cells to O6meG could be associated with a defect in MRS. The results of mismatch repair complementation studies indicated that Jurkat cells are defective in hMutSalpha, a heterodimer of the hMSH2 and hMSH6 proteins. Cytogenetic analysis of two Jurkat clones revealed a deletion in the short arm of chromosome region 2p15-21, indicating an allelic loss of both hMSH2 and hMSH6 genes. DNA sequencing revealed that exon 13 of the second hMSH2 allele contains a base substitution at codon 711, which changes an arginine to a termination codon (CGA-->TGA). In addition, a (C)8-->(C)7 frameshift mutation in codon 1085-1087 of the hMSH6 gene was also found. Although both hMSH2 and hMSH6 transcripts could be detected in Jurkat clones, the respective polypeptides were absent. Taken together, these data indicate that tolerance of Jurkat cells to methylation damage is linked to a loss of functional hMutSalpha.