The proteasome as a druggable target with multiple therapeutic potentialities: Cutting and non-cutting edges.

Ubiquitin Proteasome System (UPS) is an adaptable and finely tuned system that sustains proteostasis network under a large variety of physiopathological conditions. Its dysregulation is often associated with the onset and progression of human diseases; hence, UPS modulation has emerged as a promising new avenue for the development of treatments of several relevant pathologies, such as cancer and neurodegeneration. The clinical interest in proteasome inhibition has considerably increased after the FDA approval in 2003 of bortezomib for relapsed/refractory multiple myeloma, which is now used in the front-line setting. Thereafter, two other proteasome inhibitors (carfilzomib and ixazomib), designed to overcome resistance to bortezomib, have been approved for treatment-experienced patients, and a variety of novel inhibitors are currently under preclinical and clinical investigation not only for haematological malignancies but also for solid tumours. However, since UPS collapse leads to toxic misfolded proteins accumulation, proteasome is attracting even more interest as a target for the care of neurodegenerative diseases, which are sustained by UPS impairment. Thus, conceptually, proteasome activation represents an innovative and largely unexplored target for drug development. According to a multidisciplinary approach, spanning from chemistry, biochemistry, molecular biology to pharmacology, this review will summarize the most recent available literature regarding different aspects of proteasome biology, focusing on structure, function and regulation of proteasome in physiological and pathological processes, mostly cancer and neurodegenerative diseases, connecting biochemical features and clinical studies of proteasome targeting drugs.

Sox2 is not required for melanomagenesis, melanoma growth and melanoma metastasis in vivo.

Melanoma is a dangerous form of skin cancer derived from the malignant transformation of melanocytes. The transcription factor SOX2 is not expressed in melanocytes, however, it has been shown to be differentially expressed between benign nevi and malignant melanomas and to be essential for melanoma stem cell maintenance and expansion in vitro and in xenograft models. By using a mouse model in which BRafV600E mutation cooperates with Pten loss to induce the development of metastatic melanoma, we investigated if Sox2 is required during the process of melanomagenesis, melanoma growth and metastasis and in the acquisition of resistance to BRAF inhibitors (BRAFi) treatments. We found that deletion of Sox2 specifically in Pten null and BRafV600E-expressing melanocytes did not prevent tumor formation and did not modify the temporal kinetics of melanoma occurrence compared to Sox2 wt mice. In addition, tumor growth was similar between Sox2 wt and Sox2 deleted (del) melanomas. By querying publicly available databases, we did not find statistically significant differences in SOX2 expression levels between benign nevi and melanomas, and analysis on two melanoma patient cohorts confirmed that Sox2 levels did not significantly change between primary and metastatic melanomas. Melanoma cell lines derived from both Sox2 genotypes showed a similar sensitivity to vemurafenib treatment and the same ability to develop vemurafenib resistance in long-term cultures. Development of vemurafenib resistance was not dependent on SOX2 expression also in human melanoma cell lines in vitro. Our findings exclude an oncogenic function for Sox2 during melanoma development and do not support a role for this transcription factor in the acquisition of resistance to BRAFi treatments.

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.

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.

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.

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.

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

Cytotoxic and clastogenic effects of a DNA minor groove binding methyl sulfonate ester in mismatch repair deficient leukemic cells.

Mismatch repair deficiency contributes to tumor cell resistance to O6-guanine methylating compounds and to other antineoplastic agents. Here we demonstrate that MeOSO2(CH2)2-lexitropsin (Me-Lex), a DNA minor groove alkylating compound which generates mainly N3-methyladenine, has cytotoxic and clastogenic effects in mismatch repair-deficient leukemic cells. Moreover, MT-1 cells, which express p53 upon drug treatment and possess low levels of 3-methylpurine DNA glycosylase activity, are more susceptible to cytotoxicity induced by Me-Lex, with respect to p53-null and 3-methylpurine DNA glycosylase-proficient Jurkat cells. In both cell lines, the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide, which inhibits base excision repair capable of removing N-methylpurines, increases cytotoxicity and clastogenicity induced by Me-Lex or by temozolomide, which generates low levels of N3-methyl adducts. The enhancing effect is more evident at low Me-Lex concentrations, which induce a level of DNA damage that presumably does not saturate the repair ability of the cells. Nuclear fragmentation induced by Me-Lex + 3-aminobenzamide occurs earlier than in cells treated with the single agent. Treatment with Me-Lex and 3-aminobenzamide results in augmented expression of p53 protein and of the X-ray repair cross-complementing 1 transcript (a component of base excision repair). These results indicate that N3-methyladenine inducing agents, alone or combined with poly(ADP-ribose) polymerase inhibitors, could open up novel chemotherapeutic strategies to overcome drug resistance in mismatch repair-deficient leukemic cells.

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.

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.

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.

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.

Involvement of the mismatch repair system in temozolomide-induced apoptosis.

Postreplicative mismatch repair plays a major role in mediating the cytotoxicity of agents generating O6-methylguanine in DNA. We previously showed that a methylating antitumor triazene compound, temozolomide, induces apoptosis and that the persistence of O6-methylguanine in DNA is required to trigger the process. We wanted to test whether the latter apoptotic signal is dependent on a functional mismatch repair system. To this end, we used two human lymphoblastoid cell lines (i.e., the mismatch repair-proficient TK6 line and its mismatch repair-deficient subline MT1) that are both deficient in O6-methylguanine repair. Temozolomide treatment of TK6 cells brought about efficient cell growth inhibition, G2/M arrest, and apoptosis, as indicated by the results of cytofluorimetric analysis of 5-bromo-2'-deoxyuridine incorporation and DNA content and evaluation of DNA fragmentation. The drug treatment resulted also in the induction of p53 and p21/waf-1 protein expression. In contrast, MT1 cells were highly resistant to the drug and no p53 and p21/waf-1 induction was observed. Importantly, we could show that MT1 cells are not deficient in the p53-dependent apoptosis pathway; treatment with etoposide, a topoisomerase II inhibitor, resulted in p53 and p21/waf-1 protein expression and apoptosis in both cell lines. In conclusion, we demonstrate the existence of a link between a functional mismatch repair system and the trigger of apoptosis in cells exposed to clinically relevant concentrations of temozolomide. The results also suggest that p53 induction in response to O6-guanine methylation involves the mismatch repair system.

Role of wild-type p53 on the antineoplastic activity of temozolomide alone or combined with inhibitors of poly(ADP-ribose) polymerase.

The DNA repair enzyme O6-alkylguanine DNA-alkyltransferase (OGAT) and a deficient mismatch repair system play a critical role in the resistance to chemotherapeutic agents that generate adducts at the O6-position of guanine. However, DNA adducts different from O6-methylguanine might be also involved in cytotoxicity induced by methylating agents. Because the loss of p53 function is generally associated with tumor cell resistance to anticancer chemotherapy, we have investigated whether wild-type p53 might affect chemosensitivity of leukemia cells endowed with high OGAT levels to the methylating agent temozolomide (TZM). The effect of poly(ADP-ribose) polymerase (PADPRP) inhibition, which potentiates the cytotoxic effects of N7-methylguanine and N3-methylguanine, was also assessed in OGAT-proficient cells, either susceptible or tolerant to O6-methylguanine. OGAT-proficient and p53 null HL60 cells were transfected with the human p53 cDNA (p53+ cells). Treatment with TZM concentrations not toxic for the cells transduced with the control vector (p53-cells), induced apoptosis in p53+ cells. These cells were characterized by a lower level of bcl-2 protein than p53- cells, whereas bax and OGAT expression was comparable in both lines. Inhibition of PADPRP potentiated the cytotoxic and apoptotic effects of TZM in either p53- or p53+ HL60 cells. Furthermore, PADPRP inhibitors potentiated apoptosis induced by TZM in Jurkat cells, which possess a mutated p53 gene and are tolerant to O6-methylguanine adducts. The analysis of cell cycle indicated that the drug combination of TZM and PADPRP inhibitors provoked G1 arrest only in p53+ cells. Conversely, G1 arrest was not observed in p53+ cells exposed to TZM alone. It is possible to speculate that PADPRP inhibitors might affect the repair of DNA adducts that are processed differently from O6 methylguanine and induce a different pattern of cell cycle distribution. In conclusion, the results show that p53 increases apoptosis by TZM in OGAT-proficient cells and suggest the potential role of PADPRP inhibitors in enhancing TZM activity against leukemias independently of DNA repair systems.

Inhibition of O6-alkylguanine DNA-alkyltransferase or poly(ADP-ribose) polymerase increases susceptibility of leukemic cells to apoptosis induced by temozolomide.

High levels of expression of the DNA repair enzyme O6-alkylguanine DNA-alkyltransferase (OGAT) (EC 2.1.1.63) account for tumor cell resistance to methylating agents. Previous studies suggested that methylating triazenes might have a potential role for the treatment of acute leukemias with low levels of OGAT. In the current study, we transduced the human OGAT cDNA in OGAT-deficient leukemia cell clones. OGAT-transduced cells were more resistant than their OGAT-deficient counterparts to apoptosis triggered by the methylating triazene temozolomide (TZM), as indicated by the results of flow cytometry, terminal deoxynucleotidyl transferase assay, and analysis of DNA fragmentation. Depletion of OGAT activity by O6-benzylguanine increased leukemia cell sensitivity to TZM-mediated apoptosis. Moreover, combined treatment of cells with TZM and benzamide, an inhibitor of the poly(ADP-ribose) polymerase (EC 2.4.2.30), increased the apoptosis induced by the methylating agent. These results demonstrate for the first time that methyl adducts at the O6 position of guanine, which are specifically removed by OGAT, are the principal DNA lesions responsible for the induction of apoptosis on treatment of leukemic cells with the methylating triazene TZM. This study also supports the possible use of TZM for the treatment of acute leukemias and suggests new strategies to increase the susceptibility of tumor cells to methylating triazenes in the clinic.

In vitro inactivation of human O6-alkylguanine DNA alkyltransferase by antitumor triazene compounds.

The cytotoxic and mutagenic properties of antitumor triazene compounds (TZC) have been mainly attributed to their ability to form DNA adducts at the O6 position of guanine. Repair of these lesions is mediated by O6-alkylguanine DNA alkyltransferase (OGAT) in an autoinactivating reaction. Therefore when lesion repair has occurred, cells are depleted of OGAT until synthesis of new enzyme molecules takes place. In this study, we have evaluated the ability of DNA alkylated by different TZC to deplete OGAT activity. Moreover, we have also investigated whether these compounds might inactivate the OGAT enzyme by a direct reaction with the protein. Human OGAT protein was partially purified from insect cells infected with a recombinant baculovirus containing the human OGAT coding sequences. Thereafter human OGAT protein was exposed directly to TZC or to TZC-alkylated DNA. Among the TZC tested, p-(3-methyl-1-triazeno)benzoic acid was the most effective OGAT inactivator by direct interaction with the protein. Moreover DNA substrates treated with methylating TZC, such as temozolomide or p-(3-methyl-1-triazeno)benzoic acid, were more effective in depleting the repair enzyme, compared to DNA pretreated with the chloroethylating TZC mitozolomide. In conclusion, our results show that TZC inactivate in vitro OGAT activity by either direct or indirect mechanisms. Therefore TZC are good candidates for 1) increasing their own cytotoxicity, if used according to appropriate dose and treatment schedules and 2) reversing tumor cell resistance to O6-guanine alkylating agents.

Triazene compounds induce apoptosis in O6-alkylguanine-DNA alkyltransferase deficient leukemia cell lines.

Previous studies demonstrated that triazene compounds (TZC) possess antitumor, antimetastatic and immunosuppressive activity, and induce novel antigenic properties in neoplastic cells. Moreover, TZC showed marked antitumor activity in patients with acute myelogenous leukemias (AML). In most cases leukemic blasts with low levels of the repair enzyme O6-alkyl-guanine-DNA alkyltransferase (OGAT) were highly susceptible to TZC. Therefore the cytotoxic effects of TZC against human leukemic cells and the influence of OGAT modulation were investigated. Five leukemia cell lines were treated with the in vitro active derivative of dacarbazine: 5-(3-methyl-1-triazeno) imidazole-4-carboxamide (MTIC), or with temozolomide (TZM), which is readily cleaved to form the linear triazene MTIC in aqueous solution. The results showed that treatment with TZC at concentrations ranging between 62.5 and 250 microM significantly inhibited cell growth of U-937 and K-562 leukemia cell lines, both with undetectable OGAT activity. Growth inhibition was accompanied by DNA fragmentation and reduction of cell volume characteristic of cell undergoing apoptosis. In contrast, Daudi, HL-60 and Jurkat leukemia cell lines, characterized by high levels of the repair enzyme, were resistant to concentrations of TZC up to 500 microM. Treatment of resistant lines with O6-benzylguanine (BG, a specific inhibitor of OGAT) rendered HL-60 and Daudi but not Jurkat cells sensitive to cytotoxic effects and apoptosis mediated by MTIC. The results presented suggest that: (1) apoptosis is involved in cytotoxic activity of TZC; (2) OGAT could have a role in preventing programmed cell death induced by TZC; and (3) treatment with BG could potentiate cytotoxic and apoptotic effects of TZC on leukemic cell lines when high level of OGAT activity is the main factor involved in resistance to TZC.

Effects of cyclopentenone prostaglandins on myeloid cells during early infection with HTLV-I. I. Cell differentiation determines sensitivity to prostaglandins and virus infection.

Human myeloid cell lines at different stages of differentiation (K562, HL60 and U937) were used to analyze the permissivity of the myelomonocytic lineage to acute infection with human T-cell leukemia virus type-I (HTLV-I) after cell-to-cell transmission and to evaluate the effect of cyclopentenone prostaglandins (PG)A1 and PGJ2 on virus transmission, proliferation of recipient cells and cell-mediated cytotoxicity against virus-donor cells. Exposure to HTLV-I delayed the growth rate of recipient cells, especially in U937 cells. This effect was related to the phase of cell cycle when cells were exposed to HTLV-I. Treatment of control and virus-exposed cells with these PGs, both inducing growth arrest prevalently at the G1/S interphase of the cell cycle, inhibited cell proliferation in a concentration-dependent way. The antiproliferative effect of both PGs increased progressively from pluripotent K562 to promyelocytic HL60 and monoblastoid U937 cells, suggesting that differentiated cells were more susceptible to PG-mediated inhibition of growth than pluripotent cells. PG treatment influenced the permissivity of recipient cells to HTLV-I, with different effects on less differentiated myeloid cells in comparison with more differentiated monoblastoid cells. In fact, the percentage of cells positive for the p19gag protein was increased among PG-treated K562 or HL60 cells, although it was reduced in PG-treated U937 cells. To this respect, PGA1 was more effective on asynchronous and PGJ2 on synchronous U937 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cyclopentenone prostaglandins on myeloid cells during early infection with HTLV-I. II. Regulation of synthesis of inducible p72 heat shock protein.

Differentiation of cells of myelomonocitic lineage influences both cellular permissivity to infection with human T-cell leukemia virus type I after cell-to-cell virus transmission and sensitivity to the antiproliferative effect of cyclopentenone prostaglandins (PG)A1 and PGJ2. Growth inhibition and control of infection were found to be associated with high intracellular levels of inducible p72 heat shock protein (HSP70). Pluripotent K562 cells produced higher HSP70 base-line levels than promyelocytic HL60 or monoblastoid U937 cells. Treatment with PGA1 and especially with PGJ2 enhanced the synthesis of HSP70 in all these cells. Notably, HSP70 accumulated in virus-exposed U937 cells (but not in K562 or HL60 cells). Because in lethally irradiated virus-donor cells HSP70 production was barely detectable, expression of this protein in cocultured U937 cells can be prevalently attributed to virus-recipient cells. Treatment with PGA1 and even more with PGJ2 remarkably enhanced the synthesis of HSP70 in virus-exposed U937 cells, thus resulting in persistently high levels of HSP70 protein in the cells. As shown previously, in U937 cells treatment with PGs was associated with reduced percentages of virus p19gag positive cells and enhanced specific lysis of virus-donor cells at early time points after cell-to-cell transmission. Because the HSP70 protein family is involved in the control of cell proliferation as well as in antigen processing function during the immune response to pathogens, it is possible that persistent high expression levels of HSP70 in PG-treated cells play a critical role in regulating both cell cycling and antiviral cellular responses.