Biomedical subjects

B Kaina

Publications and source records attributed to B Kaina.

At least 37 records · Page 2Linked to original sources

PubMed2001

Acquired resistance of melanoma cells to the antineoplastic agent fotemustine is caused by reactivation of the DNA repair gene MGMT.

Acquired resistance to antineoplastic agents is a frequent obstacle in tumor therapy. Malignant melanoma cells are particularly well known for their unresponsiveness to chemotherapy; only about 30% of tumors exhibit a transient clinical response to treatment. In our study, we investigated the molecular mechanism of acquired resistance of melanoma cells (MeWo) to the chloroethylating drug fotemustine. Determination of O(6)-methylguanine-DNA methyltransferase (MGMT) activity showed that MeWo cells that acquired resistance to fotemustine upon repeated treatment with the drug display high MGMT activity, whereas the parental cell line had no detectable MGMT. The resistant cell lines exhibit cross-resistance to other O(6)-alkylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine. Acquired resistance to fotemustine was alleviated by treatment with the MGMT inhibitor O(6)-benzylguanine demonstrating that reactivation of MGMT is the main underlying cause of acquired alkylating drug resistance. As compared with control cells, both MGMT mRNA and MGMT protein were expressed at a high level in fotemustine resistant cells. Southern blot analysis proved that the MGMT gene was not amplified. There was also only an insignificant difference in the CpG methylation pattern of the MGMT promoter whereas a clear hypermethylation in the body of the gene was observed in fotemustine resistant cells. The conclusion that hypermethylation is responsible for reactivation of the MGMT gene gained support by the finding that MGMT activity significantly declined and cells reverted (partially) to the parental sensitive phenotype upon treatment with 5-azacytidine. This is the first report of acquired resistance to a chloroethylating antineoplastic drug of melanoma cells due to gene hypermethylation.

Antineoplastic Agents↗

PubMed2001

Metallothionein expression in ovarian cancer in relation to histopathological parameters and molecular markers of prognosis.

Metallothioneins (MTs) and glutathione constitute the major fractions of intracellular thiol factors. Abundant nucleophilic sulfhydryl groups can interact with many electrophilic substances, including several anti-neoplastic agents, participate in controlling intracellular redox potential, and act as scavengers of reactive oxygen species. In the present study, we examined the relation of MTs (alone and in combination with glutathione) to histopathological parameters and survival time of ovarian cancer patients. Expression of the major MT isoforms (MT-1 and MT-2) was determined by immunohistochemistry on paraffin-embedded tumor specimens from 189 patients, 151 suffering from primary epithelial ovarian cancer and 38 from recurrences. MT was negatively associated with survival time when all patients with primary carcinomas (n = 151) were analyzed (p = 0.049, log-rank test). However, no significant association between MT expression and survival was obtained when subgroups of patients with histological grade 1, 2 or 3 carcinomas were analyzed. Similarly, no significant association of MT expression and survival was obtained with the proportional hazards model adjusted for histological grade. This scenario can be explained by a correlation between MT expression and histological grade: MT was detectable in 26%, 48% and 62% of grade 1, 2 and 3 carcinomas, respectively (p = 0.008, chi(2) test). An interesting hypothesis is generated by combined analysis of MT and total glutathione content (GSH). The product of MT and GSH levels (MT x GSH) was negatively associated with survival of grade 1 carcinomas (p = 0.021, log-rank test) but not with grade 2 and 3 carcinomas (p = 0.176 and 0.403, respectively). When MT x GSH was greater than the median, 25% of patients with grade 1 carcinomas died within 235 days. In contrast, all patients with grade 1 carcinomas survived when MT x GSH in tumor tissue was smaller than the median. This suggests that high expression of sulfhydryl factors might facilitate survival and progression of low-grade ovarian cancer cells. A significant correlation was obtained between MT expression and mutant p53 (p = 0.037, chi(2) test). However, this might be an indirect effect since both MT (p = 0.008) and mutant p53 (p = 0.000) were associated with histological grade. In conclusion, MT expression as well as the product of MT and GSH were associated with histological grade of primary ovarian carcinomas. High expression of both sulfhydryl factors may identify a subgroup of low-grade carcinomas with an increased risk of progression.

Disease Progression↗

PubMed2001

Hamster Bcl-2 protein is cleaved in vitro and in cells by caspase-9 and caspase-3.

Full-length cDNA of hamster bcl-2 (771 nt) was cloned by RT-PCR and inserted into pGEX-4T-1 to produce the recombinant hamster Bcl-2 protein. The purified recombinant Bcl-2 protein (26.4 kDa) was used as a substrate for the active human caspase-3 and caspase-9 in vitro. It is shown here that Bcl-2 is efficiently cleaved by caspase-3 to a 23 kDa fragment. Although not possessing a putative caspase-9 cleavage site in its sequence, hamster Bcl-2 was also cleaved by caspase-9 into exactly the same 23 kDa cleavage product, indicating that cleavage occurred at the same site. Caspase-3- and caspase-9-mediated cleavage of Bcl-2 was efficiently blocked by caspase-3 (zDEVD) and caspase-9 (zLEHD) inhibitor, respectively. We also show that caspase-9/-3-mediated cleavage of Bcl-2 occurs in vivo during apoptosis in CHO-HSV-TK cells after exposure to the antiviral drug ganciclovir.

Animals↗

PubMed2001

Transcriptional activation of the small GTPase gene rhoB by genotoxic stress is regulated via a CCAAT element.

The gene encoding the Ras-related GTPase RhoB-specific is immediate-early inducible by genotoxic treatments. Regulation of transcriptional activation of rhoB is still unclear. Here we show that cells lacking either p53 or c-Fos are not different from wild-type cells with respect to the level of rhoB induction upon UV irradiation, indicating that these transcription factors are not crucial for stimulation of rhoB mRNA expression. Extracts from UV-irradiated and non-irradiated cells revealed similar DNA-binding activities to a 0.17 kb rhoB promoter fragment harboring the functional element(s) necessary for stimulation of rhoB by UV light. By means of immunoprecipitation we found that an ATF-2-specific antibody co-precipitates the (32)P-labeled 0.17 kb rhoB fragment, whereas an anti-AP1 antibody did not. Since no consensus sequence for binding of ATF-2 is present within the rhoB promoter, ATF-2 is likely to be associated with another factor that binds to the minimal promoter. Deletion analysis and site-directed mutagenesis of the 0.17 kb rhoB fragment revealed a CCAAT box to be an essential requirement for stimulation of rhoB by UV light and methyl methanesulfonate. Moreover, immunoprecipitation experiments showed that the CCAAT-binding factor NF-YA is complexed with ATF-2. Overall, the data strongly indicate that transcriptional activation of the rhoB gene by genotoxic stress is regulated via a CCAAT box and that interaction of CCAAT-binding factor and ATF-2 triggers the stress-inducible expression of rhoB.

3T3 Cells↗

PubMed2001

Long-time expression of DNA repair enzymes MGMT and APE in human peripheral blood mononuclear cells.

The DNA repair enzymes O6-methylguanine-DNA methyltransferase (MGMT) and apurinic/apyrimidinic endonuclease (APE, also known as Ref-1) play an important role in cellular defense against the mutagenic and carcinogenic effects of DNA-damaging agents. Cells with low enzyme activity are more sensitive to induced DNA damage and may confer a higher carcinogenic risk to the individuals in question. To study the level of variability of MGMT and APE expression in human, we analyzed in a long-time study MGMT and APE expression in peripheral blood mononuclear cells (PBMC) from healthy individuals. The data revealed high inter- and intraindividual variability of MGMT but not of APE. For MGMT, the interindividual levels ranged from 27 to 204 fmol/10(6) cells (7.6-fold, 40 healthy individuals). The intraindividual variation was determined by measuring MGMT repeatedly over 42 days, and was found to vary from 1.4-fold to 3.5-fold. Averaging over the measurement period, some individuals displayed low MGMT activity compared to others. In contrast, APE expression showed only a 2.9-fold difference between individuals and a 1.2 to 2.3-fold intra-individual long-time variation, and thus was less variable than MGMT. MGMT and APE expression were not correlated. Overall the results showed variable MGMT and rather constant APE levels in PBMC of healthy individuals measured over a long period.

Adult↗

PubMed2001

BER, MGMT, and MMR in defense against alkylation-induced genotoxicity and apoptosis.

Methylating carcinogens and cytostatic drugs induce different methylation products in DNA. In cells not expressing the repair protein MGMT or expressing it at a low level, O6-methylguanine is the major genotoxic, recombinogenic, and apoptotic lesion. Genotoxicity and apoptosis triggered by O6-methylguanine require mismatch repair (MMR). In cells expressing O6-methylguanine-DNA methyl transferase (MGMT) at a high level or for agents producing low amounts of O6-methylguanine, N-alkylations become the major genotoxic lesions. N-Alkylations are repaired by base excision repair (BER). In mammalian cells, naturally occurring mutants of BER have not been detected, which points to the importance of BER for viability. In order to ascertain the role of BER in cellular defense, BER was modulated either by transfection or mutational inactivation. It has been shown that overexpression of N-methylpurine-DNA glycosylase (MPG) does not protect, but rather sensitizes cells to SN2 agents. This has been interpreted in terms of an imbalance in BER. Regarding abasic site endonuclease (APE), transient but not stable overexpression of the enzyme was achieved upon transfection in CHO cells, which indicates that unphysiologic APE levels are not tolerated by the cell. Besides the repair function, APE (alias Ref-1) exerts redox capability by which the activity of various transcription factors is modulated. Therefore, it is possible that stable overexpression of mammalian APE impairs transcriptional regulation of genes, whereas transient overexpression may exert some protective effect. DNA polymerase beta (Pol beta) transfection was ineffective in conferring resistance to methylmethane sulfonate (MMS). On the other hand, Pol beta-deficient cells proved to be highly sensitive to methylation-induced chromosomal aberrations and reproductive cell death. The dramatic hypersensitivity in the killing response is largely due to induction of apoptosis. Obviously, nonrepaired BER intermediates are clastogenic and act as a strong trigger of the apoptotic pathway. The elements of this pathway are currently under investigation.

Alkylation↗

PubMed2001

DNA double-strand breaks trigger apoptosis in p53-deficient fibroblasts.

DNA double-strand breaks (DSBs) are induced by ionizing radiation (IR) and various radiomimetic agents directly, or indirectly as a consequence of DNA repair, recombination and replication of damaged DNA. They are ultimately involved in the generation of chromosomal aberrations and were reported to cause genomic instability, gene amplification and reproductive cell death. To address the question of whether DSBs act as a trigger of apoptosis, we induced DSBs by means of restriction enzyme electroporation and compared the effect with IR in mouse fibroblasts that differ in p53 status [wild-type (+/+) versus p53-deficient (-/-) cells]. We show that (i) electroporation of PVU:II is highly efficient in the induction of DSBs, (ii) electroporation of PVU:II increases the rate of apoptosis, but not of necrosis in p53-/- cells, (iii) treatment with gamma-rays induces both apoptosis and necrosis in p53-/- cells, (iv) the frequency of DSBs correlates with the yield of apoptosis and (v) both PVU:II and gamma-ray treatment reduce the level of anti-apoptotic Bcl-2 protein in p53-/- cells whereas the level of Bax remains unaltered. Cells expressing wild-type p53 were more resistant than p53-deficient cells as to the induction of apoptosis and did not show Bcl-2 decline upon treatment with PVU:II and gamma-rays. The data provide evidence that blunt-ended DSBs induced by restriction enzyme PVU:II act as a highly efficient trigger of apoptosis, but not of necrosis. This process is related to Bcl-2 decline and does not require p53.

Animals↗

PubMed2001

Effect of ultraviolet light, methyl methanesulfonate and ionizing radiation on the genotoxic response and apoptosis of mouse fibroblasts lacking c-Fos, p53 or both.

c-Fos and p53 are DNA damage-inducible proteins that are involved in gene regulation, cell cycle checkpoint control and cell proliferation following exposure to genotoxic agents. To investigate comparatively the role of c-Fos and p53 in the maintenance of genomic stability and the induction of apoptosis, we generated mouse fibroblast cell lines from knockout mice deficient for either c-fos (fos -/-) or p53 (p53-/-) or for both gene products (fosp53-/-). The sensitivity of these established cell lines was compared with the corresponding wild-type cells as to the cytotoxic, clastogenic and apoptosis-inducing effects of ultraviolet (UV-C) light and methyl methanesulfonate (MMS). Additionally, we analysed the frequency of apoptosis of the cell lines after treatment with ionizing radiation (IR). We observed c-fos-/-, p53-/- and fosp53-/- cells to be more sensitive than wild-type cells with respect to cell death, as measured in a cytotoxicity (MTT) assay. Regarding apoptosis, all deficient cell lines displayed hypersensitivity to UV-C light, MMS and IR. With chromosomal aberrations as the endpoint, the sensitivity of the double-knockout cells was between wild-type and single-knockouts. The results indicate that both c-Fos and p53 play an important role in protecting fibroblasts against a broad range of genotoxic agents. The results also show that, in fibroblasts, apoptosis induced by UV-C light, MMS and IR does not require p53 and that, in this cell type, p53 rather protects against DNA damage-induced apoptotic cell death.

Animals↗

PubMed2000

Primary mouse fibroblasts deficient for c-Fos, p53 or for both proteins are hypersensitive to UV light and alkylating agent-induced chromosomal breakage and apoptosis.

The important regulatory proteins, c-Fos and p53 are induced by exposure of cells to a variety of DNA damaging agents. To investigate their role in cellular defense against genotoxic compounds, we comparatively analysed chromosomal aberrations and apoptosis induced by ultraviolet (UV-C) light and the potent alkylating agent methyl methanesulfonate (MMS) in primary diploid mouse fibroblasts knockout for either c-Fos or p53, or double knockout for both genes. We show that c-Fos and p53 deficient fibroblasts are more sensitive than the corresponding wild-type cells as to the induction of chromosomal aberrations and apoptosis. Double knockout fibroblasts lacking both c-Fos and p53 are viable and were even more sensitive, showing additivity of the chromosomal breakage effects observed in the single knockouts. Regarding the endpoint apoptosis, double knockout fibroblasts displayed a sensitivity similar to c-Fos and p53 deficient cells. The data indicate that (a) both c-Fos and p53 are involved in cellular protection against the clastogenic effect of genotoxic agents, (b) p53 is not required for induction of apoptosis by UV light and MMS, but rather prevents fibroblasts from undergoing apoptotic cell death upon DNA damage, and (c) c-Fos and p53 seem to act independently in determining genotoxic resistance, which is hypothesized to be achieved by impaired DNA repair or differential cell cycle check point control.

Alkylating Agents↗

PubMed2000

Nuclear translocation of mismatch repair proteins MSH2 and MSH6 as a response of cells to alkylating agents.

Mammalian mismatch repair has been implicated in mismatch correction, the prevention of mutagenesis and cancer, and the induction of genotoxicity and apoptosis. Here, we show that treatment of cells specifically with agents inducing O(6)-methylguanine in DNA, such as N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea, elevates the level of MSH2 and MSH6 and increases GT mismatch binding activity in the nucleus. This inducible response occurs immediately after alkylation, is long-lasting and dose-dependent, and results from translocation of the preformed MutSalpha complex (composed of MSH2 and MSH6) from the cytoplasm into the nucleus. It is not caused by an increase in MSH2 gene activity. Cells expressing the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), thus having the ability to repair O(6)-methylguanine, showed no translocation of MutSalpha, whereas inhibition of MGMT by O(6)-benzylguanine provoked the translocation. The results demonstrate that O(6)-methylguanine lesions are involved in triggering nuclear accumulation of MSH2 and MSH6. The finding that treatment of cells with O(6)-methylguanine-generating mutagens results in an increase of MutSalpha and GT binding activity in the nucleus indicates a novel type of genotoxic stress response.

Active Transport, Cell Nucleus↗

PubMed2000

Ras-related GTPase Rhob represses NF-kappaB signaling.

rhoB encoding a Ras-related GTPase is immediate-early inducible by genotoxic treatments, indicating that it is part of the cellular stress response. Here, we investigated the influence of RhoB on signal pathways that are rapidly evoked by genotoxic compounds. The data obtained show that wild-type RhoB neither affects activation of mitogen-activated protein kinases nor AP-1-dependent gene expression. However, RhoB inhibited both basal and genotoxic agent-stimulated activity of the transcription factor nuclear factor kappaB (NF-kappaB). Thus, RhoB attenuated alkylation-induced increase in the DNA binding activity of NF-kappaB and abrogated NF-kappaB-driven gene expression. Furthermore, RhoB inhibited decrease in the cellular amount of IkappaBalpha after genotoxic stress as well as after tumor necrosis factor alpha and 12-O-tetradecanoylphorbol acetate treatment. This indicates that RhoB represses NF-kappaB activation by inhibiting dissociation and subsequent degradation of IkappaBalpha. On the basis of the data, we suggest that RhoB is a novel negative regulator of NF-kappaB signaling.

3T3 Cells↗

PubMed2000

Apoptosis induced by DNA damage O6-methylguanine is Bcl-2 and caspase-9/3 regulated and Fas/caspase-8 independent.

In the therapy of various kinds of tumors, methylating agents generating O6-methylguanine (O6MeG) in DNA are used. We studied the molecular mechanism of cell death induced by these agents by comparing isogenic cell lines proficient (MGMT+) and deficient (MGMT-) for the DNA repair protein alkyltransferase and exhibiting the tolerance phenotype. Hypersensitivity to methylation-induced cell killing of MGMT- cells is attributable to the potent induction of apoptosis. We show that apoptosis is a late event occurring >48 h after methylation. It was preceded by decrease in Bcl-2 protein level and accompanied by activation of caspase-9 and caspase-3. We also observed cytochrome c release and hypophosphorylation of Bad. Other members of the Bcl-2 family (Bag-1, Bak, Bax, and Bcl-xL) were not altered in expression. Transfection of MGMT- cells with bcl-2 protected against methylation-induced apoptosis, indicating that Bcl-2 plays a key role in the response. Induction of apoptosis in MGMT- cells was not triggered by Fas and Fas ligand (CD95, Apo-1) because both proteins remained unaltered in expression and receptor-proximal caspase-8 was not activated after methylation. Also, inhibition of caspase-8 was ineffective in modifying the apoptotic response, whereas inhibition of caspase-3 and caspase-9 blocked apoptosis. Tolerant cells that are unable to repair O6MeG and are impaired in mismatch repair were less sensitive regarding the induction of apoptosis and Bcl-2 decline, supporting the view that O6MeG-induced apoptosis requires mismatch repair. The ultimate O6MeG-derived lesions triggering the apoptotic pathway are likely to be DNA double-strand breaks, which were significantly formed in MGMT- but not in MGMT+ and tolerant cells and which preceded apoptosis. Overall, the data indicate that O6MeG induces apoptosis via secondary lesions that trigger Bcl-2 decline, cytochrome c release, and caspase-9 and caspase-3 activation independently of Fas/Fas ligand and p53, for which the cells are mutated.

Animals↗

PubMed2000

Cloning and functional analysis of cDNA encoding the hamster Bcl-2 protein.

We have cloned cDNA encoding hamster Bcl-2 protein from total RNA of CHO-9 cells by RT-PCR using oligonucleotide primers sharing homology with the sequence of mouse and rat bcl-2. The fragments spanning the total coding region were cloned into pCR4-TOPO and sequenced for verification. The hamster bcl-2 cDNA has a size of 711 nucleotides and encodes a polypeptide of 236 amino acids. Hamster Bcl-2 shares 95.8 and 88.6% similarity with mouse and human Bcl-2, respectively. Northern blot analysis revealed a single 7.5 kb bcl-2 transcript in hamster (CHO-9), mouse (BK4), and rat (H5) cells and a 8.5 kb bcl-2 mRNA in human (HeLa MR) cells. The bcl-2 cDNA (771 bp) was recloned into pcDNA3 and the recombinant construct was transiently transfected into MGMT-deficient HeLa MR cells. Expression of hamster Bcl-2 rendered the cells more resistant to MNNG-induced cytotoxicity, which is consistent with the anti-apoptotic function of Bcl-2.

Alkylation↗

PubMed2000

Ras-related GTPase RhoB forces alkylation-induced apoptotic cell death.

rhoB encoding a Ras-related GTPase is immediate-early inducible by genotoxic treatments. To address the question of the physiological role of RhoB in cellular defense, cells stably overexpressing wild-type RhoB protein were generated. Overexpression of RhoB renders cells hypersensitive to the killing effect of alkylating agents including antineoplastic drugs but not to UV-light and doxorubicin. As compared to control cells, RhoB overexpressing cells revealed an increase in the frequency of alkylation-induced apoptotic cell death. This indicates that RhoB is involved in modulating apoptotic signaling. Furthermore, overexpression of RhoB resulted in a prolonged transient block to DNA replication upon MMS treatment. UV-induced replication blockage was not affected by RhoB. Based on the data we suggest RhoB to be a novel regulatory factor which takes influence on the level of cytotoxicity of DNA damaging drugs and forces cells to alkylation-induced apoptosis. The data indicate that this might be due to RhoB mediated delay in cell cycle progression upon alkylation treatment.

3T3 Cells↗

PubMed2000

Comparison of the genotoxic and apoptosis-inducing properties of ganciclovir and penciclovir in Chinese hamster ovary cells transfected with the thymidine kinase gene of herpes simplex virus-1: implications for gene therapeutic approaches.

We studied the genotoxic and apoptosis-inducing properties of ganciclovir (GCV) and penciclovir (PCV) using Chinese hamster ovary cells stably transfected with the thymidine kinase (tk) gene of herpes simplex virus-1 (HSV-1). Cells expressing HSVtk were 300 and 100 times more sensitive than their isogenic HSVtk- counterparts to the cytotoxic effects of GCV and PCV, respectively. Using radiolabeled drugs, GCV was found to be incorporated into the genomic DNA much more effectively than PCV. GCV was highly potent in inducing chromosomal aberrations compared with PCV, which provoked less sister chromatid exchanges and chromosomal changes using equimolar or equitoxic doses. For both agents, apoptosis was shown to be the major route of cell killing. Time course experiments revealed that neither genotoxicity nor apoptosis were induced within the cell cycle exposed to the drug; they are late events provoked in the following cell cycle(s). This indicates that the incorporation/exposure step of GCV or PCV into DNA is not decisive for triggering genotoxicity and apoptosis, but that events occurring subsequently, presumably during replication of a DNA containing the nucleotide analogs, are of major importance. Because PCV, unlike GCV, induced highly effectively apoptosis without exerting much genotoxicity, the use of PCV as a relatively safe alternative drug for suicide gene therapy of malignant diseases is recommended.

Acyclovir↗

PubMed2000

Mechanistic aspects of the cytotoxic activity of glufosfamide, a new tumour therapeutic agent.

Beta-D-glucosyl-ifosfamide mustard (D 19575, glc-IPM, INN = glufosfamide) is a new agent for cancer chemotherapy. Its mode of action, which is only partly understood, was investigated at the DNA level. In the breast carcinoma cell line MCF7 glufosfamide inhibited both the synthesis of DNA and protein in a dose-dependent manner, as shown by the decreased incorporation of [3H-methyl]-thymidine into DNA and [14C]-methionine into protein of these cells. Treatment of MCF7 cells with 50 microM glufosfamide was sufficient to trigger poly(ADP-ribose) polymerase (PARP) activation, as revealed by immunofluorescence analysis. Both CHO-9 cells, which are O6-methylguanine-DNA methyltransferase (MGMT)-deficient, and an isogenic derivative, which has a high level of MGMT, showed the same cytotoxic response to beta-D-glc-IPM, indicating that the O6 position of guanine is not the critical target for cytotoxicity. By contrast, a sharp decrease in survival of cross-link repair deficient CL-V5 B cells was observed already at concentrations of 0.1 mM beta-D-glc-IPM, whereas the wild-type V79 cells showed a 90% reduction in survival only after treatment with 0.5 mM of this compound. The therapeutically inactive beta-L-enantiomer of glufosfamide also showed genotoxic effects in the same assays but at much higher doses. This was probably due to small amounts of ifosfamide mustard formed under the conditions of incubation. The results indicate that the DNA crosslinks are the most critical cytotoxic lesions induced by beta-D-glc-IPM.

Animals↗

PubMed2000

Induction of heme oxygenase-1 and adaptive protection against the induction of DNA damage after hyperbaric oxygen treatment.

Hyperbaric oxygen (HBO) treatment of human subjects (i.e. exposure to 100% oxygen at a pressure of 2.5 ATA for a total period of 3 x 20 min) caused clear and reproducible DNA damage in lymphocytes, as detected with the comet assay (single cell gel electrophoresis). Induction of DNA damage was found only after the first HBO exposure and not after further treatments of the same individuals. Furthermore, blood taken 24 h after HBO treatment was significantly protected against the induction of DNA damage by hydrogen peroxide (H(2)O(2)) in vitro, indicating that adaptation occurred due to induction of antioxidant defenses. The cells were not significantly protected against the genotoxic effects of gamma-irradiation, suggesting increased scavenging of reactive oxygen species distant from nuclear DNA or an inducible change in the levels of free transition metals. We now demonstrate increased levels of heme oxygenase-1 (HO-1) in lymphocytes 24 h after HBO treatment of volunteers. Under the same conditions, superoxide dismutase, catalase and the DNA repair enzymes apurinic endonuclease and DNA polymerase beta were not enhanced in expression. We also show that protection against the induction of DNA damage by H(2)O(2) in lymphocytes even occurs with a shortened HBO treatment which did not induce significant DNA damage by itself. Our results suggest that increased sequestration of iron as a consequence of induced HO-1 might be involved in the adaptive protection after HBO treatment and that the induction of DNA damage is not the trigger for adaptive protection.

Adaptation, Physiological↗

PubMed2000

Cytogenetic genotoxicity of anti-herpes purine nucleoside analogues in CHO cells expressing the thymidine kinase gene of herpes simplex virus type 1: comparison of ganciclovir, penciclovir and aciclovir.

The three anti-herpes nucleoside analogues ganciclovir, penciclovir and aciclovir were investigated as to their recombinogenic [sister chromatid exchange (SCE) inducing] and clastogenic activity in CHO cells expressing the thymidine kinase gene of HSV-1, which is a precondition of therapeutic activity of these drugs. The compounds were applied for the duration of one cell cycle and cytogenetic end-points were measured between 0 and 42 h after exposure. Although the nucleoside analogues are quite similar with respect to chemical structure, they differ basically in their genotoxic potency, aberration types induced as well as the time course of chromosomal damage. Aciclovir induced SCEs and chromosomal aberrations immediately after exposure but only in a concentration range much higher than that reached in blood plasma during anti-herpes therapy. The direct genotoxic activity is explained by the obligate chain terminating property of aciclovir upon incorporation into genomic DNA. On the other hand, genotoxic damage caused by ganciclovir and penciclovir is of the delayed type requiring at least one post-exposure cell cycle for its expression. Unlike aciclovir, ganciclovir is an extremely potent inducer of SCEs and chromosome breaks and translocations at concentrations far below those impairing the proliferative activity and triggering apoptosis of the target cells (as shown by our previous investigation). Penciclovir is essentially devoid of genotoxic activity. It induces SCEs only at cytotoxic/apoptotic concentrations, is only weakly clastogenic and induces premature chromosome condensation which appears to result from uncoupling of karyokinesis and cytokinesis. The genotoxic activity of ganciclovir is explained as due to repair processes triggered in the second post-exposure replication cycle at the sites of nucleoside analogue incorporation into genomic DNA. The findings have considerable implications with respect to the use of ganciclovir or other antiviral drugs in suicide gene therapy of malignant diseases.

Acyclovir↗