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.

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.

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.

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.

Inhibition of protein isoprenylation impairs rho-regulated early cellular response to genotoxic stress.

Activation of c-Jun N-terminal kinases (JNKs) and nuclear factor-kappaB (NF-kappaB) are early cellular responses to genotoxic stress involved in the regulation of gene expression. Pretreatment of cells with the hydroxymethyl glutaryl-CoA reductase inhibitor lovastatin blocked stimulation of JNK1 activity by UV irradiation and by treatment with the alkylating compound methyl methanesulfonate but did not affect activation of extracellular signal-regulated kinase 2 by UV light. Lovastatin also attenuated UV-induced degradation of the NF-kappaB inhibitor IkappaBalpha.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Overexpression of cDNA encoding FANCC, SPHAR, MPG, SNM1 or HA 3611 does not render CHO cells more resistant to DNA crosslinking agents.

DNA crosslinking agents (DCA) are commonly used cytostatic drugs, whose efficiency in tumor therapy is limited due to the appearance of drug resistant tumor cells. In an effort to modulate the resistance of cells to DCA, we transfected into Chinese hamster cells various cDNAs whose loss of function was previously shown to render cells more sensitive to crosslinking agents. We show that overexpression of FANCC, SPHAR, MPG, SNM1 or HA 3611 (a human homologue of the yeast crosslink DNA repair gene SNM1) does not alter the level of resistance of CHO cells to clinically relevant DCA, such as mafosfamide, melphalan and mitomycin C.

Cell and tissue-type specific expression of Ras-related GTPase RhoB.

Ras-homologous (Rho) GTPases are involved in the regulation of a variety of cellular processes such as the organization of the actin cytoskeleton, malignant transformation and genotoxic stress-induced signaling. Here we show that, among the family of Rho GTPases, specifically rhoB mRNA expression is rapidly induced upon UV-irradiation, whereas the level of rac 1 and cdc42 mRNA is not affected. Increase in rhoB mRNA was accompanied by a approximately 4-fold increase in the amount of membrane-bound RhoB protein.

Transcriptional activation of apurinic/apyrimidinic endonuclease (Ape, Ref-1) by oxidative stress requires CREB.

Apurinic/apyrimidinic endonuclease (APE alias Ref-1) is a multifunctional enzyme involved in DNA repair and redox regulation of transcription factors (e.g., AP-1).

Activity of O(6)-methylguanine-DNA methyltransferase in relation to p53 status and therapeutic response in ovarian cancer.

The DNA-repair protein O(6)-methylguanine-DNA methyltransferase (alkyltransferase; MGMT) is a major determinant of resistance of cells to various alkylating cytostatic drugs. Its expression in tissues is highly variable, indicating complex regulatory mechanisms involved. Transfection-mediated expression of wild-type p53 has been shown to negatively regulate basal promoter activity of MGMT in vitro.

Rho GTPases are over-expressed in human tumors.

Small GTPases of the Rho family are involved in the regulation of a variety of cellular processes, such as the organization of the microfilamental network, cell-cell contact and malignant transformation. To address the question of whether Rho proteins are involved in carcinogenesis in man, we compared their expression in tumors from colon, breast and lung with that of the corresponding normal tissue originating from the same patient. As shown by Rho-specific 32P-ADP-ribosylation, as well as Western-blot analysis, the amount of RhoA protein was largely increased in all 3 types of tumors tested.

Cells deficient in DNA polymerase beta are hypersensitive to alkylating agent-induced apoptosis and chromosomal breakage.

DNA polymerase beta (beta-pol), which is involved in base excision repair, was investigated for its role in protection of cells against various genotoxic agents and cytostatic drugs using beta-pol knockout mouse fibroblasts. We show that cells lacking beta-pol are highly sensitive to induction of apoptosis and chromosomal breakage by methylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine and methyl methanesulfonate and the cross-linking antineoplastic drugs mitomycin C and mafosfamide. The cross-sensitivity between the agents observed suggests that beta-pol is involved in repair not only of DNA methylation lesions but also of other kinds of DNA damage induced by various cytostatic drugs.

Expression of DNA repair proteins hMSH2, hMSH6, hMLH1, O6-methylguanine-DNA methyltransferase and N-methylpurine-DNA glycosylase in melanoma cells with acquired drug resistance.

Malignant melanoma is well known for its primary unresponsiveness to chemotherapy. The mechanisms conferring this intrinsic resistance are unclear. In this study, we investigated the role of genes involved in DNA repair in a panel of human melanoma cell variants exhibiting low and high levels of resistance to 4 commonly used drugs in melanoma treatment, i.

Phosphorylation of the DNA repair protein APE/REF-1 by CKII affects redox regulation of AP-1.

The DNA repair protein apurinic endonuclease (APE/Ref-1) exerts several physiological functions such as cleavage of apurinic/apyrimidinic sites and redox regulation of the transcription factor AP-1, whose activation is part of the cellular response to DNA damaging treatments. Here we demonstrate that APE/Ref-1 is phosphorylated by casein kinase II (CKII). This was shown for both the recombinant APE/Ref-1 protein (Km=0.

Activation of c-Jun N-terminal kinase 1 by UV irradiation is inhibited by wortmannin without affecting c-iun expression.

Activation of c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases is an early response of cells upon exposure to DNA-damaging agents. JNK-mediated phosphorylation of c-Jun is currently understood to stimulate the transactivating potency of AP-1 (e.g.

Nickel(II) inhibits the repair of O6-methylguanine in mammalian cells.

Nickel compounds are widespread carcinogens, and although only weakly mutagenic, interfere with nucleotide excision repair and with the repair of oxidative DNA base modifications. In the present study we investigated the effect of nickel(II) on the induction and repair of O6-methylguanine and N7-methylguanine after treatment with N-methyl-N-nitrosourea (MNU). We applied Chinese hamster ovary cells stably transfected with human O6-methylguanine-DNA methyltransferase (MGMT) cDNA (CHO-AT), and compared the results with the MGMT-deficient parental cell line.

p53 is involved in regulation of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) by DNA damaging agents.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is inducible by genotoxic stress. MGMT induction results from transcriptional activation of the MGMT gene which is a specific response to DNA damage. A possible factor involved in triggering MGMT induction might be p53, because both p53 and MGMT are activated by DNA breaks.