Epigenetic regulation of the MGMT and hMSH6 DNA repair genes in cells resistant to methylating agents.

We investigated the relationship between DNA cytosine methylation and the expression of two genes associated with resistance to DNA methylation damage. Variants of RajiMex- cells acquired resistance to N-methyl-N-nitrosourea by either reactivating a previously silent O6-methylguanine-DNA methyltransferase (MGMT) gene or by repressing the hMSH6 mismatch repair gene. DNA sequencing and measurements of mRNA and enzyme levels revealed that MGMT activity was not correlated with methylation of the core MGMT promoter. Treatment with the demethylating agent 5-azadeoxycytidine reduced MGMT mRNA and enzyme levels, indicating that methylation of some nonpromoter sequences may be required for MGMT gene expression. In contrast, both hMSH6 mRNA and protein levels were increased by 5-azadeoxycytidine treatment of an N-methyl-N-nitrosourea-resistant variant that did not express detectable hMSH6, which implies that this gene was transcriptionally silenced by cytosine methylation. This could be substantiated by in vitro modification of the CpG sites in the hMSH6 promoter with restriction methylase M.SssI, which abolished the transcription of a reporter gene under its control in a transient transfection assay. Taken together, our data show that treatment with chemical methylating agents alters gene expression patterns through increased CpG methylation of genomic DNA, and thereby permits the emergence and selection of clones that are resistant to these agents due to increased repair or tolerance of O6-methylguanine.

Effect of melphalan and hyperthermia on cell cycle progression and cyclin B1 expression in human melanoma cells.

We have investigated the effect of mild hyperthermia (42 degrees C) on the cytotoxic activity of a 1 h melphalan exposure in human melanoma cell lines. Hyperthermia did not affect cell growth of any culture, but it increased, to a different extent, melphalan cytotoxicity in all cell lines, with a reduction in the IC50 of 1.7 to 2.6-fold. Flow cytometric analysis showed that in normal temperature conditions melphalan caused S phase cell accumulation, which was evident only at 24 h in JR8, M14 and 2/21 cell lines and was still persistent at 72 h in 2/60 cells. Moreover, in all cell lines, the delay in S phase was paralleled, or followed, by an accumulation of cells in G2+M, which was transient in JR8 and M14 cells and persisted until 72 h in 2/21 and 2/60 melanoma clones. Hyperthermia caused a stabilization and prolongation of melphalan induced G2+M accumulation in JR8 and M14 cells. Conversely, in 2/21 and 2/60 clones, cell cycle perturbations induced by the drug were similar under normothermic or hyperthermic conditions. Specifically, in JR8, for which the maximum enhancement by hyperthermia on melphalan cytotoxicity was observed, cell accumulation in G2+M was still present 120 h after treatment. The accumulation was accompanied by an inhibition in the G2-M transition, as demonstrated by the significant reduction in the mitotic index of cells exposed to combined treatment compared to controls. Moreover, a bivariate distribution of cells stained for DNA and cyclin B1 showed that, following melphalan and hyperthermia treatment, the fraction of cyclin B1-expressing cells paralleled the fraction of G2+M phase cells, thus indicating that the inability of cells to enter mitosis was not ascribable to a reduction of cyclin B1 expression. On the whole, our results indicate that hyperthermia can stabilize the G2 accumulation induced by melphalan in human melanoma cells. Such a stabilization could contribute to the enhancement of melphalan cytotoxicity by heat, even though a strict correlation was not observed between the magnitude and persistence of the cell cycle perturbations and the extent of melphalan activity.

Effects of a novel trinuclear platinum complex in cisplatin-sensitive and cisplatin-resistant human ovarian cancer cell lines: interference with cell cycle progression and induction of apoptosis.

We evaluated the effects of the trinuclear platinum complex, BBR 3464, in a human ovarian carcinoma cell line (OAW42) and in its cisplatin (CDDP)-resistant counterpart (OAW42MER). A 14-fold increased sensitivity to a 1-h BBR 3464 exposure was found in OAW42MER cells compared with their parental cell line. Flow cytometric experiments showed that BBR 3464 was able to induce a persistent block of OAW42 and OAW42MER cells in the G2M phase, whereas CDDP caused an initial accumulation of cells in the S phase followed by an increase in the G2M cell fraction in both cell lines. Exposure to equitoxic (IC(50)) drug concentrations induced programmed cell death in both cell lines. However, the percentage of cells with an apoptotic nuclear morphology was slightly higher after CDDP than BBR 3464 treatment in OAW42 cells, whereas the opposite pattern was observed in OAW42MER cells. Degradation of the nuclear lamin B was detected in OAW42 cells after exposure to each drug. Conversely, in OAW42MER cells lamin B cleavage was only appreciable after BBR 3464 exposure. In OAW42 cells, CDDP and BBR 3464 did not appreciably affect the mitochondrial membrane potential (Deltapsi(mt)), whereas in the OAW42MER cell line a marked Deltapsi(mt) reduction was observed after exposure to BBR 3464, but not to CDDP. The results of the study would suggest that the sensitivity to BBR 3464 observed in the CDDP-resistant OAW42MER cell line might be attributable to the ability of the trinuclear platinum complex to modify DNA in a way which is different from that of CDDP and, as a consequence, to induce different cellular responses to DNA damage such as the triggering of specific apoptotic pathways.

In vitro effect of lonidamine on the cytotoxicity of mitomycin C and BCNU in human colon adenocarcinoma cells.

The ability of lonidamine (LND), an energolytic derivative of indazole carboxylic acid, to modulate the cytotoxic activity of mitomycin C (MMC) and 1,3,bis(2-chloroethyl)-1-nitrosourea (BCNU) was investigated in two human adenocarcinoma cell lines (LoVo and HT29) expressing different sensitivity profiles to the drugs. After a 1-h treatment with MMC or BCNU, cells were postincubated for 24 h with 150-225 microM LND. In the LoVo cells, a synergistic interaction between LND and MMC or BCNU was observed at both LND concentrations. In HT29 cells, only additive effects of the drugs given in sequence were seen. Flow cytometric analysis indicated that LND was generally able to stabilise the cell cycle perturbations induced by MMC and BCNU in the two cell lines. The ability of LND to potentiate anticancer drug activity, and the consideration that LND causes side-effects different from those of conventional antitumour drugs, make this compound an attractive candidate for multidrug combination therapy in colon cancer.

Nimodipine as a modulator of resistance to doxorubicin in human colon-adenocarcinoma cells.

The potential of the dihydropyridine nimodipine (NIMO), a drug currently used for cerebrovascular disorders, in modulating the cytotoxicity of doxorubicin (DX) was investigated, in comparison to verapamil (VER), in human colon adenocarcinoma cell lines sensitive (LoVo) or resistant (LoVo/DX) to anthracycline. NIMO selectively enhanced DX activity in the resistant cell line. In particular, the DX concentration able to inhibit cell growth by 50% was reduced by 2.6 to 5.7-fold as a function of the time of exposure to the modulator. The increase in DX accumulation in LoVo/DX nuclei after exposure to NIMO was about 4-fold that observed in the nuclei of resistant cells exposed to DX alone. Similar results were obtained in LoVo/DX cells following treatment with equimolar concentrations of VER. Again, both NIMO and VER failed to interfere with the immunoreactivity of LoVo/DX cells to the MRK16 monoclonal antibody. NIMO did not inhibit H-3-azidopine binding to LoVo/DX cells, but, unlike VER, it caused a selective hyperpolarization of the cell membrane in resistant cells.

Cell growth on cordierite: an approach to the identification of reliable supports for continuous-flow solid-bed reactors.

This study aimed to assess the biocompatibility of two cordierite ceramics (DF and Cord 1014), with similar chemical composition and different porosity, as a potential support for cell growth in a continuous-flow, solid-bed reactor. The Chinese hamster ovary (CHO) cell line transfected with HBV-DHFR recombinant plasmid was seeded on cordierite or polystyrene dishes and evaluated for cell growth and production of recombinant hepatitis B surface antigen. Proliferation of the CHO cells, in terms of cell number, was generally similar in polystyrene and Cord 1014 and always lower in DF. Flow cytometric analysis showed no difference in cell cycle distribution for cells grown on different supports, and showed a two-fold increase in percentage of debris for cells grown on DF than for those grown on Cord 1014 and polystyrene culture dishes. Moreover, the morphology of cells grown on Cord 1014 did not change during the experiment, and cells were well spread and organized. Finally, total recombinant hepatitis B surface antigen production was higher on Cord 1014 than on polystyrene and DF samples. Such evidence suggests that Cord 1014 could be a promising support for growing cells in a continuous-flow, solid-bed reactor.

Combined effects of interferon-alpha2a and 13-cis-retinoic acid on human melanoma cell growth and STAT protein expression.

We assessed the antiproliferative effects of human recombinant interferon-alpha2a (IFNalpha2a) and 13-cis-retinoic acid (13cis-RA) on two human melanoma cell lines (JR8 and M14). Both cell lines showed a very modest sensitivity to IFNalpha2a and 13cis-RA as single agents. In JR8 cells, the combination of the two compounds consistently produced simple additive effects. In contrast, different effects of the combination were recorded in the M14 cell line depending on the treatment schedule. Specifically, an additive interaction was observed when IFNalpha2a and 13cis-RA were given in sequence, independently of the order of drug administration, whereas a supra-additive antiproliferative effect was seen when cells were simultaneously exposed to the two drugs. Exposure to 1000 IU/ml IFNalpha2a markedly increased the nuclear expression of signal transducers and activators of transcription (STAT) proteins in both cell lines. By itself 10 microM 13cis-RA did not affect STAT protein expression or modify the extent of activation of such proteins by IFNalpha2a. Results from our study showed an enhancement of the antiproliferative activity of IFNalpha2a and 13cis-RA when given in combination and suggest that such an enhancement is not mediated by a concomitant effect of 13cis-RA on STAT protein activation.

Lack of p21waf1 and p27kip1 protein induction by interferon-alpha2a in human melanoma cell lines.

The ability of human recombinant interferon-alpha2a (IFNalpha2a) to induce the expression of cyclin-dependent kinase inhibitors p21waf1 and p27kip1 consequent to signal transducers and activators of transcription (STAT) protein activation was investigated in six human melanoma cell lines with different susceptibilities to the antiproliferative effect of the cytokine. All the cell lines expressed IFNalpha and IFNalpha/beta receptors. Exposure for 24 h to IFNalpha2a markedly enhanced the nuclear expression of STAT1 and STAT2 proteins in all the cell lines. However, no induction of p21waf1 or p27kip1 was consistently observed. Overall, results from the study suggest that the induction of such cyclin-dependent kinase inhibitors is not a major mechanism for the antiproliferative effect of IFNalpha2a, at least in human melanoma cell lines.

DNA double-strand break repair and radiation response in human tumour primary cultures.

The accumulation and repair of radiation-induced DNA double-strand breaks (dsbs) were determined by neutral filter elution on 20 primary cultures obtained from ovarian cancer and malignant melanoma clinical specimens. The initial frequency of DNA dsbs after exposure to 50 Gy gamma-irradiation varied greatly for the individual cultures. However, melanomas were generally more efficient than ovarian cancers in repairing these DNA lesions (mean percentage of DNA dsb rejoined after 2 h: 83 versus 62%). In 13 of 20 cultures radiosensitivity was also assessed by the Courtenay clonogenic assay. The mean +/- SD of the surviving fraction at 2 Gy (SF2) was slightly higher for melanomas (0.56 +/- 0.25) than for ovarian carcinomas (0.43 +/- 0.23). No correlation was observed between SF2 and in vitro plating efficiencies or any biological characteristics of the tumour cell population, such as proliferative activity and DNA ploidy. Similarly, we failed to find any relation between the initial frequencies of DNA dsbs and SF2 in individual tumours. In contrast, a significant and direct relationship (r = 0.86, p < 0.01) was observed between SF2 and the percentages of DNA dsbs rejoined 2 h after irradiation. In agreement with reported data on human tumour established cell lines, our results indicate that the ability to repair DNA dsbs is an important determinant for radiation response even in primary cultures of clinical tumours.

Effect of melphalan and hyperthermia on p34cdc2 kinase activity in human melanoma cells.

We previously reported that combined treatment with melphalan and mild hyperthermia (1 h at 42 degrees C) caused a synergistic cytotoxic effect in JR8 melanoma cells, paralleled by a stabilisation of a melphalan-induced G2-phase cell block. In this study, we investigated the effect of melphalan and hyperthermia on proteins that regulate G2-M transition. Neither hyperthermia nor melphalan at a concentration of 2.5 micrograms ml-1, which had no antiproliferative effect at 37 degrees C, interfered with cyclin B1 expression or p34cdc2 kinase activity. At a concentration of 8.5 micrograms ml-1, which reduced cell growth by 50% at 37 degrees C, melphalan inhibited p34cdc2 kinase activity as a consequence of an increased tyrosine phosphorylation of the protein. A similar inhibitory effect on p34cdc2 kinase was obtained when the lowest melphalan concentration (2.5 micrograms ml-1) was used under hyperthermic conditions. Our results indicate that thermal enhancement of melphalan cytotoxicity could be mediated at least in part by an inhibition of p34cdc2 kinase activity, which prevents cell progression into mitosis.

Modulation by lonidamine on the combined activity of cisplatin and epidoxorubicin in human breast cancer cells.

The ability of lonidamine (LND), an energolytic derivative of indazole-carboxylic acid, to modulate the cytotoxic activity of cisplatin (CDDP) and epidoxorubicin (EPI), singly or in combination, was investigated in two human breast cancer cell lines (MCF7 and T47D). A 72-hr post-incubation with a non-cytotoxic concentration of LND (75 microM) increased the activity of a 1-hr CDDP treatment as well as that of a 1 to 16-hr EPI treatment. A different pattern of interaction among the drugs and modulator was observed as a function of the sequence of drug treatment. Specifically, supra-additive or additive effects of the combination were obtained in the two cell lines according to the different treatment schemes. In particular, the maximum potentiation was observed in MCF7 cells simultaneously exposed to CDDP, EPI and LND for 1 hr and then post-incubated with LND for 72 hr, and in T47 first exposed to EPI and LND, then to CDDP and LND, and finally post-incubated with LND. Flow cytometric analysis of MCF7 cell distribution in the different cycle phases showed that combined treatment with EPI/CDDP/LND was able to stabilize cell cycle perturbations (mainly G2M accumulation) induced by individual agents. The ability of LND to potentiate CDDP and EPI cytotoxicity, and the consideration that LND causes side effects different from those caused by alkylating agents and anthracyclines, make this compound an attractive candidate for multidrug combination therapy in breast cancer.

Lack of a correlation between p53 protein expression and radiation response in human tumor primary cultures.

We investigated the possible relationship between immunohistochemically detected p53 expression and in vitro response to gamma-irradiation in 24 primary cultures of human ovarian cancers and cutaneous melanomas. The frequency of p53-positive tumors was around 60% within each tumor histotype. The range of the surviving fraction at 2 Gy (SF2) was similar in p53-positive (0.10-0.76) and p53-negative (0.23-0.65) tumors, with median values of 0.36 and 0.33, respectively. No differences were observed in the accumulation of DNA-double strand breaks, assessed by neutral filter elution after exposure to 50 Gy, between p53-positive and p53-negative tumors. As regards DNA lesion repair, after 2 h of recovery the percentage of rejoined DNA-double strand breaks ranged from 19% to 99% in the different cultures, but again the distribution of values was similar for p53-positive and p53-negative tumors. Specifically, the median percentage of repaired DNA-double strand breaks was 70% and 74% in the two groups. On the whole, our data do not support the hypothesis that p53 overexpression is a major determinant of in vitro radiation response.

Induction of apoptosis by taxol and cisplatin and effect on cell cycle-related proteins in cisplatin-sensitive and -resistant human ovarian cells.

The effect of taxol (TX) and cisplatin (CDDP), singly or in association, was assessed on two human ovarian cancer cell lines, one sensitive (A2780) and one resistant (A2780 cp8) to CDDP. Cell lines showed a similar sensitivity to TX, whereas different cytotoxicity results were obtained in the two cell lines as a function of TX and CDDP sequence. Specifically, TX followed by CDDP induced simply additive effects in both cell lines, whereas the opposite sequence produced antagonistic effects in A2780 cells and synergistic effects in A2780 cp8 cells. TX, with or without CDDP, induced oligonucleosomal DNA fragmentation typical of the apoptotic process, but the biochemical mechanisms undergoing apoptosis were different in the two cell lines. In fact, in A2780 cells, TX (with or without CDDP) treatment markedly increased p53 as well as p21waf1 protein expression. In A2780 cp8 cells, drug treatment enhanced p53 levels, whereas the expression of p21waf1 was always undetectable at mRNA and protein levels. In the latter cell line, a premature activation of p34cdc2 kinase was observed in correspondence with the drug-induced increase in the S-phase cell fraction. Such an activation was not ascribable to an increase in the overall expression of p34cdc2 or cyclin B1 proteins, but to a dephosphorylation of p34cdc2 kinase. Overall, our results indicate that TX-induced apoptosis in human ovarian cancer cells may be sustained by different events at the cell cycle-control level.

Lonidamine as a modulator of taxol activity in human ovarian cancer cells: effects on cell cycle and induction of apoptosis.

The ability of lonidamine (LND), an energolytic derivative of indazole-carboxylic acid, to modulate the cytotoxicity of Taxol (TX) was investigated in the A2780 human ovarian cancer cell line. Different cytotoxicity results were obtained as a function of treatment schedule. Specifically, TX followed by LND produced synergistic effects. Conversely, antagonistic effects were recorded when drugs were given simultaneously or according to the opposite sequence. TX induced an oligonucleosomal DNA fragmentation typical of the apoptotic process. The extent and the kinetics of DNA cleavage in samples treated with the taxane alone were similar to those of samples treated with the TX-LND sequence. Activation of Yama protease and degradation of poly (ADP-ribose) polymerase were not observed after individual or combined treatment. LND did not appreciably modify the effect exerted by TX on proteins involved in cell cycle progression (i.e., inhibition of p34cdc2 expression) and apoptosis (i.e., upregulation of wt p53 and transactivation of p21waf1), and only caused a slight induction of the Bax protein. LND alone did not affect tubulin polymerization in A2780 cells and, when administered after a 24 hr TX exposure, did not appreciably alter the extent of tubulin polymerization induced by the taxane. Although additional studies are needed to define the molecular basis of the TX-LND interaction, our results suggest that LND can positively modulate the antitumor activity of TX in ovarian cancer cells and indicate that the energolytic is potentially useful in combination therapy including the taxane in ovarian cancer patients.

Effect of ionizing radiation on cell-cycle progression and cyclin B1 expression in human melanoma cells.

In the present study we investigated the effect of gamma-irradiation (2.5 and 10 Gy) on cell-cycle progression of a human melanoma cell line, M14, characterized by a moderate radiosensitivity (SF2 = O.5). Flow cytometric analysis showed a dose-dependent S-phase accumulation, which was detectable 8 hr after treatment with 2 and 5 Gy and was still persistent at 12 hr after 10 Gy exposure. Such a delay in S-phase was paralleled or followed by an accumulation of cells in G2M, which was transient at the lowest radiation doses and still persistent at 72 hr after 10 Gy. Such an accumulation was, at least in part, due to a block in G2-M transition, as demonstrated by mitotic index analysis. Bivariate flow cytometric analysis of DNA content and cyclin B1 expression showed that, following 2 and 5 Gy, the fraction of cyclin B1-expressing cells was superimposable upon that of G2M cells. Conversely, in cells treated with 10 Gy, the fraction of cyclin B1-expressing cells was half the G2M cell fraction. Northern-blot analysis indicated that the radiation-induced decrease in cyclin B1 protein expression was accompanied by a reduced cyclin B mRNA level. On the whole, our results indicate a direct inhibitory effect of 10 Gy irradiation on cyclin B1 expression as a possible cause for the persistent G2 block in irradiated M14 cells.

Involvement of bcl-2 and p21waf1 proteins in response of human breast cancer cell clones to Tomudex.

Mechanisms of resistance to Tomudex include increased thymidylate synthase activity, as well as reduced intracellular drug uptake and polyglutamation. However, little is known about other mechanisms of resistance, such as a possible protection against Tomudex-induced apoptosis mediated by bcl-2. We transfected the MDA-MB-435 human breast cancer cell line, which is characterized by a mutated p53 gene, with cDNA of the bcl-2 gene and generated two clones (MDA-bcl4 and MDA-bcl7) characterized by bcl-2 expression twofold and fourfold that observed in the control cell clone (MDAneo). A concomitant overexpression of p21wafl was also detected in the MDA-bcl7 clone. The MDA-bcl4 clone was three times more resistant to a 24-h Tomudex exposure than the MDAneo clone, whereas the MDA-bcl7 clone was as sensitive to Tomudex as the control cell clone. A lower sensitivity of the MDA-bcl4 clone than MDAneo and MDA-bcl7 clones to 5-fluorouracil and gemcitabine was also observed. No significant difference was noted in the susceptibility of clones to fludarabine and methothrexate. Basal levels of thymidylate synthase activity were superimposable in the three clones. Tomudex induced a marked accumulation of cells in the S phase in all the clones. However, an apoptotic hypodiploid DNA peak and the characteristic nuclear morphology of apoptosis were observed only in the MDA-bcl7 clone after exposure to Tomudex. No difference in the treatment-induced modulation of proteins involved in cell cycle progression (cyclin A, cdk2, pRB, E2F-1) and apoptosis (bcl-2, bax) was observed in the three clones. The only exception was that the expression of p21wafl in the MDA-bcl4 clone was inducible at a Tomudex concentration much higher than that required to induce the protein in the other clones. Overall, the results indicate that bcl-2 and p21wafl proteins concur in determining the cellular profile of sensitivity/resistance to Tomudex.

Activity of a trinuclear platinum complex in human ovarian cancer cell lines sensitive and resistant to cisplatin: cytotoxicity and induction and gene-specific repair of DNA lesions.

A collateral sensitivity or a very modest cross-resistance to BBR 3464 was found in 2 ovarian cancer cell lines with experimentally induced resistance to cisplatin. Loss of mismatch repair proteins (hMLH1, hPMS2) or overexpression of nucleotide excision repair proteins (ERCC1) was not detrimental for the cellular sensitivity to BBR 3464. Moreover, interesting differences in the kinetics of formation and removal of DNA lesions at the single-gene (N- ras) level were observed between BBR 3464 and CDDP.