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A V Gudkov

Publications and source records attributed to A V Gudkov.

At least 37 records · Page 2Linked to original sources
PubMed2000

Suppression of p53: a new approach to overcome side effects of antitumor therapy.

The p53 protein is traditionally believed to be a tumor suppressor. Activation of p53-dependent apoptosis in response to damage to cell DNA provides for the elimination of possible tumor cell precursors. However, in some cases the activity of p53 can be dangerous for the organism. Thus, p53-dependent apoptosis induced in normal tissues during chemo- and radiotherapy can cause severe side effects of antitumor therapy and, therefore, limits its efficiency. This review analyzes experimental data on the role of p53 in the primary and late tissue response to DNA-damaging exposures. Comparison of normal and p53-deficient mice indicated that the apoptosis in radiosensitive tissues during the first hours after irradiation is really caused by the activity of p53 which, in turn, is determined by a high level of expression of mRNA of p53. We supposed that a temporary suppression of p53 can decrease the damage to sensitive tissues and accelerate their recovery after the antitumor radio- and chemotherapy. To test this hypothesis, we have isolated a chemical inhibitor of p53 and determined its activity in vitro and in vivo. This compound, called pifithrin-alpha, protects wild-type mice against lethal doses of radiation, has no effect on p53-deficient animals, and does not induce visible tumors. These results show that the suppression of p53 is a promising approach in the prevention of side effects of antitumor therapy.

Animals↗
PubMed1999

Structure and regulation of the mouse ing1 gene. Three alternative transcripts encode two phd finger proteins that have opposite effects on p53 function.

The human ING1 gene encodes nuclear protein p33(ING1), previously shown to cooperate with p53 in cell growth control (Garkavtsev, I., Grigorian, I. A., Ossovskaya, V. S., Chernov, M. V., Chumakov, P. M., and Gudkov, A. V. (1998) Nature 391, 295-298). p33(ING1) belongs to a small family of proteins from human, mouse, and yeast of approximately the same size that show significant similarity to one another within the C-terminal PHD finger domain and also contain an additional N-terminal region with subtle but reliably detectable sequence conservation. Mouse ing1 is transcribed from three differently regulated promoters localized within a 4-kilobase pair region of genomic DNA. The resulting transcripts share a long common region encoded by a common exon and differ in their 5'-exon sequences. Two transcripts are translated into the same protein of 185 amino acids, the mouse equivalent of the human p33(ING1), while the third transcript encodes a longer protein that has 94 additional N-terminal amino acids. Overexpression of the longer protein interferes with the accumulation of p53 protein and activation of p53-responsive promoters after DNA damage. Between the two products of ing1, only the longer one forms a complex with p53 detectable by immunoprecipitation. These results indicate that a single gene, ing1, encodes both p53-suppressing and p53-activating proteins that are regulated by alternative promoters.

3T3 Cells↗
PubMed1999

A chemical inhibitor of p53 that protects mice from the side effects of cancer therapy.

Chemotherapy and radiation therapy for cancer often have severe side effects that limit their efficacy. Because these effects are in part determined by p53-mediated apoptosis, temporary suppression of p53 has been suggested as a therapeutic strategy to prevent damage of normal tissues during treatment of p53-deficient tumors. To test this possibility, a small molecule was isolated for its ability to reversibly block p53-dependent transcriptional activation and apoptosis. This compound, pifithrin-alpha, protected mice from the lethal genotoxic stress associated with anticancer treatment without promoting the formation of tumors. Thus, inhibitors of p53 may be useful drugs for reducing the side effects of cancer therapy and other types of stress associated with p53 induction.

Animals↗
PubMed1999

Generation of p53 suppressor peptide from the fragment of p53 protein.

The p53 protein, encoded by a tumor suppressor gene, mediates growth arrest or apoptosis in response to a variety of stresses. p53-dependent apoptosis, occurring in several sensitive tissues after radiation or chemotherapy, is partially responsible for the side effects of cancer treatment, making p53 a potential target for therapeutic suppression. p53 function can be suppressed by the ectopic expression of p53-derived peptides, isolated earlier using functional selection of genetic suppressor elements (GSEs) from a library of randomly fragmented p53 cDNA (Ossovskaya et al. [1996]. Proc. Natl. Acad. Sci. U.S.A. 93, 10309). The potent p53-suppressing GSE, GSE56, had been used to generate in an E. coli expression system a peptide with anti-p53 activity by fusion of the GSE-encoded sequence with penetratin, a 16-amino-acid-long peptide capable of efficient translocation through cell membranes. Fusion with penetratin does not affect the anti-p53 activity of retrovirus-transduced GSE56. The fused peptide was able to attenuate p53-mediated transactivation and apoptosis when added into culture media. Interestingly, GSE56-derived peptide with no penetratin also had accumulated in the cells and showed similar, though lower, anti-p53 activity. This study provides the rationale and methodological basis for efficient generation of biologically active peptides with therapeutic potential from GSEs isolated through functional selection.

Animals↗
PubMed1999

Forward genetics in mammalian cells: functional approaches to gene discovery.

Definitive proof of function in biological systems requires genetic analysis. Only when the loss of a particular protein corresponds to the loss of a specific function can one be sure that the protein truly affects the function. Changing the pattern of gene expression through random mutagenesis or by introducing expression libraries, followed by selection of mutant or variant cells and identification of a missing or overexpressed protein, has the power to reveal or confirm the roles of specific components of signaling pathways and to provide mutant cell lines and cDNA reagents to be used in defining detailed mechanisms through structure-function analyses. These examples of forward genetics contrast with reverse genetic approaches, where the function of a known gene product is explored by knockout or replacement. Here we review a broad range of techniques that have been used to alter gene expression randomly in mammalian cells, with examples of specific discoveries that have resulted from these applications of forward genetics.

Animals↗
PubMed1998

Stress-induced secretion of growth inhibitors: a novel tumor suppressor function of p53.

p53 tumor suppressor gene controls cell response to a variety of stresses inducing growth arrest or apoptosis in damaged cells. It largely determines the sensitivity of tumor and normal cells to radiation and chemotherapy, and, therefore, defines both the efficacy and limitations of anti-cancer treatment. To determine molecular mechanisms of p53-dependent stress response in normal tissues we identified and compared the spectra of radiation-responsive genes in cells of different origin and p53 status using a cDNA array hybridization technique. The majority of genes identified were p53-dependent and cell type specific. Several of the new p53 responders encode known secreted growth inhibitory factors. This suggests that p53, in addition to its intrinsic antiproliferation activity, can cause 'bystander effect' by inducing export of growth suppressive stimuli from damaged cells to neighboring cells. Consistently, a p53-dependent accumulation of factors, which causes growth inhibitory effects in a variety of cell lines, was found after gamma irradiation in the media from established and primary cell cultures and in the urine of irradiated mice. Moreover, p53-dependent factors released by normal human fibroblasts potentiated the cytotoxic effect of a chemotherapeutic drug on co-cultivated tumor cells. This suggests a previously unknown role for normal cells in chemo- and radiation therapy of cancer.

3T3 Cells↗
PubMed1998

Altered expression of ubiquitous kinesin heavy chain results in resistance to etoposide and hypersensitivity to colchicine: mapping of the domain associated with drug response.

The motor protein kinesin is a tetramer consisting of two heavy and two light chains. Expression of an antisense RNA fragment derived from the mouse ubiquitous kinesin heavy chain (uKHC) cDNA is associated with a unique type of multidrug resistance. We analyzed the effects of retroviral transduction of the human uKHC and its derivatives on drug sensitivity of the human fibrosarcoma cell line HT1080. Surprisingly, overexpression of full-length uKHC and its variants that were deficient in the NH2-terminal motor domain produced a phenotype similar to that of antisense RNA, characterized by resistance to etoposide and collateral sensitivity to colchicine. This altered drug response, therefore, appears to be a general consequence of kinesin deregulation. The genetic suppressor element approach was applied to map the determinants of drug response in the kinesin heavy chain. A sense-oriented genetic suppressor element conferring resistance to etoposide was isolated from a retroviral library of randomly fragmented uKHC cDNA. This element encodes the last 55 amino acids of uKHC, suggesting that the COOH-terminal tail domain of uKHC is involved in the cellular drug response.

3T3 Cells↗
PubMed1998

Cell transforming genes and tumor progression: in vivo unified secondary phenotypic cell changes.

We have earlier shown that Syrian hamster cells spontaneously transformed in vitro during in vivo progression, acquire in 1 step, along with highly increased tumorigenicity, 2 new properties characterizing the [H2O2CA + tPGE(S)] phenotype, i.e., a high H2O2 catabolizing (antioxidant) activity and the ability to release PGE2 upon contact with NK cells. In contrast, RSV-SR-(v-src)-transformed cells acquire the [H2O2CA + PGE(S)] phenotype and high tumorigenicity during in vitro transformation, i.e., without preliminary in vivo selection. In the present study, the possible influence of different transforming genes on the rates of subsequent in vivo tumor progression was studied using cells in vitro transformed by SV40, BAV-3, or transduced by activated genes Ha-ras, p53, myc and bcl-2. The expression of the [H2O2CA + PGE(S)] phenotype, the extent of tumorigenic and spontaneous metastasizing activities were examined before and during in vivo cells selection in s.c. growing tumors. Our results demonstrate that: (1) after in vitro transformation all cell lines (except v-src) were negative for the expression of [H2O2CA + PGE(S)] phenotype and remained equally low-tumorigenic; (2) independently of the types of genes initially transforming the cells, in vivo tumor progression was consistently leading to the replacement of parental cells by cells expressing the [H2O2CA + PGE(S)] phenotype, to 30-200 times increased tumorigenicity and less frequently to metastasizing; (3) the time necessary for selection of cells expressing this phenotype was the same (about 180 days in vivo) for all transformants, except bcl-2; the latter reaching similar values after a significant delay. Thus, common secondary src-like phenotypic cell changes, regardless of initially cell transforming genes are necessarily selected during tumor progression in vivo.

Animals↗
PubMed1998

The candidate tumour suppressor p33ING1 cooperates with p53 in cell growth control.

The candidate tumour-suppressor gene ING1 has been identified by using the genetic suppressor element (GSE) methodology. ING1 encodes a nuclear protein, p33ING1, overexpression of which inhibits growth of different cell lines. The properties of p33ING1 suggest its involvement in the negative regulation of cell proliferation and in the control of cellular ageing, anchorage dependence and apoptosis. These cellular functions depend largely on the activity of p53, a tumour-suppressor gene that determines the cellular response to various types of stress. Here we report that the biological effects of ING1 and p53 are interrelated and require the activity of both genes: neither of the two genes can, on its own, cause growth inhibition when the other one is suppressed. Furthermore, activation of transcription from the p21/WAF1 promoter, a key mechanism of p53-mediated growth control, depends on the expression of ING1. A physical association between p33ING1 and p53 proteins has been detected by immunoprecipitation. These results indicate that p33ING1 is a component of the p53 signalling pathway that cooperates with p53 in the negative regulation of cell proliferation by modulating p53-dependent transcriptional activation.

Animals↗
PubMed1998

Could p53 be a target for therapeutic suppression?

The function of p53 has been traditionally viewed in the context of its tumor suppressor activity. In fact, the p53-dependent growth arrest and apoptosis, occurring in response to a variety of stimuli, act to protect the organism from cancer by eliminating potential tumor precursors. However, the same functions of p53 could determine severe damage of normal tissues as a consequence of genotoxic stress associated with anti-tumor therapy. This makes p53 a potential target for therapeutic suppression with the purpose of rescuing normal tissues from the side effects of cancer treatment. We analyze the accumulated information regarding the role of p53 in acute and long-term consequences of genotoxic stress in vivo. Comparison of p53 wild type and p53-deficient mice indicates that p53, in fact, determines massive apoptosis occurring shortly after gamma irradiation in radiosensitive tissues. Sites of apoptosis match the tissue-specific pattern of p53 mRNA expression indicating that p53 regulation at mRNA level is a determinant of acute radiosensitivity of tissues. In the hematopoietic system, radiation-induced death of both differentiating and stem cells strongly depends on p53, suggesting that p53 suppression would decrease damage and promote faster recovery of hematopoiesis after anti-cancer therapy. However, p53 does not effect the recovery of radiosensitive epithelia since their stem cells, in contrast to differentiating cells, die in a p53-independent manner. The validity and potential complications of therapeutic suppression of p53 in cancer treatment and under other stressful conditions are discussed in relation to the p53 functions in normal development.

Animals↗
PubMed1997

Suppression of apoptosis by bcl-2 does not prevent p53-mediated control of experimental metastasis and anchorage dependence.

Mutations in the p53 tumor suppressor gene are frequently associated with the metastatic stage of tumor progression. Inactivation of p53 was shown to promote metastasis under experimental conditions. To determine the p53 functions that are involved in the control of tumor metastasis, we compared properties of three types of transformed mouse fibroblasts: with intact p53, with p53-mediated apoptosis suppressed by bcl-2 and with p53 inactivated by dominant negative mutants. Although expression of bcl-2 blocked apoptosis in detached cells and increased tumor cell survival in the blood circulation, it was insufficient to affect the ability of p53 to cause cell cycle arrest in detached cells and suppress experimental metastasis. For the suppression of metastasis complete inactivation of p53 was required. We conclude that the apoptotic function of p53 is dispensable for the p53-dependent suppression of experimental metastasis that is presumably achieved by controlling anchorage dependence. These data provide a possible explanation to dramatic differences in values of bcl-2 and mutant p53 as prognostic markers in human cancer.

Animals↗
PubMed1997

Intracellular localization of p53 tumor suppressor protein in gamma-irradiated cells is cell cycle regulated and determined by the nucleus.

DNA damage leads to the stabilization of p53 protein and its translocation to the nucleus, resulting in activation or suppression of p53-responsive genes. However, a significant proportion of cell nuclei remain negative for p53 and p53-inducible cyclin-dependent kinase inhibitor p21waf1 after a single dose of gamma-irradiation. Quantitation of DNA content in p53-positive and -negative nuclei 4-6 h after 10 Gy of gamma-irradiation of human breast carcinoma MCF7 cells, fibrosarcoma HT1080 cells, and diploid skin fibroblasts showed that p53 and p21waf1 nuclear accumulation occurs predominantly in the G1 phase and at the beginning of the S phase of the cell cycle. The majority of the nuclei in late S phase and in G2-M phase remained p53- and p21waf1-negative. This suggests that there is a cell cycle window during which p53 can accumulate in the nucleus and activate expression of p21waf1. To determine whether cell cycle-dependent distribution of p53 is caused by cytoplasmic modifications of p53 protein or by properties of the nucleus, p53 localization was analyzed in multinucleated cells obtained by polyethylene glycol-mediated cell fusion. Dramatic differences in p53 accumulation were found among the nuclei in individual multinucleated cells. Distribution of p53-positive and -negative nuclei among the phases of the cell cycle was similar to that observed in a regular cell population. These results suggest that the observed differences in p53 accumulation in the nuclei of irradiated cells are determined by cell cycle-dependent nuclear functions. In contrast to p53, p21waf1 was equally distributed among the nuclei of multinucleated cells regardless of the stage of the cell cycle, indicating that the observed phenomenon is specific for p53.

Breast Neoplasms↗
PubMed1997

Dominant nature of the resistance to Fas- and tumor necrosis factor-alpha-mediated apoptosis in human prostatic carcinoma cell lines.

We have recently found (O. W. Rokhlin et al., Cancer Res., 57: 1758-1768, 1997) that, although Fas ligation induced apoptosis in two of six human prostatic carcinoma cell lines investigated, the apoptotic machinery involved in Fas-mediated killing is already in place in Fas-resistant cell lines. Here, we investigated Fas- and tumor necrosis factor-alpha (TNF-alpha)-mediated apoptosis in cell hybrids between resistant (DU145 and JCA1) and sensitive (ALVA31 and PC3) cell lines. All three types of hybrid cells investigated, F1(DU145 x PC3), F1(JCA1 x PC3), and F1(JCA1 x ALVA31), were found to be resistant to Fas- and TNF-alpha-mediated apoptosis at the same level as the corresponding parental resistant cell lines. These results indicate that resistance to Fas- and TNF-alpha-mediated apoptosis dominates over sensitivity in cell hybrids and suggest that resistance may be regulated by an apoptosis suppressor factor or factors acting in resistant but not in sensitive cells.

Apoptosis↗
PubMed1997

Transgenic mice with p53-responsive lacZ: p53 activity varies dramatically during normal development and determines radiation and drug sensitivity in vivo.

To analyze the involvement of p53-dependent transcriptional activation in normal development and in response to DNA damage in vivo, we created transgenic mice with a lacZ reporter gene under the control of a p53-responsive promoter. Five independent strains showed similar patterns of transgene expression. In untreated animals, lacZ expression was limited to the developing nervous system of embryos and newborn mice and was strongly decreased in the adult brain. gamma-irradiation or adriamycin treatment induced lacZ expression in the majority of cells of early embryos and in the spleen, thymus and small intestine in adult mice. Transgene expression was p53 dependent and coincided with the sites of strong p53 accumulation. The lacZ-expressing tissues and early embryos, unlike other adult tissues and late embryos, are characterized by high levels of p53 mRNA expression and respond to DNA damage by massive apoptotic cell death. Analysis of p53-null mice showed that this apoptosis is p53 dependent. These data suggest that p53 activity, monitored by the reporter lacZ transgene, is the determinant of radiation and drug sensitivity in vivo and indicate the importance of tissue and stage specificity of p53 regulation at the level of mRNA expression.

3T3 Cells↗
PubMed1997

Applications of green fluorescent protein as a marker of retroviral vectors.

The Green Fluorescent Protein (GFP) of Aequorea victoria is used as a vital fluorescent tag for the detection and isolation of genetically modified cells. Several modified variants of GFP were tested as marker genes in retroviral vectors containing different backbones and promoter combinations. Constructs allowing for reliable detection of GFP fluorescence and the expression of a cotransduced gene from a strong promoter were identified. Cells harboring such constructs are detectable by flow cytometry, fluorescence microscopy and multi-well fluorescence reading. GFP expression in transduced cells is stable both in vitro and in vivo, and long-term dynamics of GFP-positive fractions in a mixed population can be used to monitor the biological effects of a cotransduced gene. Selection of cells with the highest GFP fluorescence enriches for multiply infected cells. The use of different GFP variants allows one to monitor simultaneously two cell populations transduced with vectors carrying GFPs that differ in their fluorescence intensity or spectral properties and to identify doubly transduced cells. In addition, transcription of an inducible promoter positioned in the opposite orientation to GFP can be monitored by the inhibition of GFP fluorescence. Thus, GFP provides a useful marker for gene transfer by retroviral vectors and extends the range of applications for retroviral transduction.

Animals↗
PubMed1997

Localization of the candidate tumor suppressor gene ING1 to human chromosome 13q34.

A novel gene ING1 was recently cloned and defined as a candidate tumor suppressor gene. Reduced expression and rearrangements of ING1 are found in several tumor cell lines, ING1 overexpression is associated with cell growth arrest and ING1 suppression promotes neoplastic transformation (1). Using radiation hybrid mapping technique ING1 was assigned to subtelomeric region of the long arm of human chromosome 13 (13q34) which is known to be frequently rearranged in squamous carcinomas of head and neck.

Animals↗
PubMed1996

p53 modulation of anchorage independent growth and experimental metastasis.

Death in circulation is one of the natural barriers preventing dissemination of tumor cells and formation of metastases. One of the negative factors acting in circulation is the loss of cell contact with natural substrate which can be imitated in vitro by the incubation of cells in suspension or in semi-solid media. Normal mouse fibroblasts (MEFs) stay viable in suspension and undergo p53-independent G1 growth arrest. Transformation with Ela and ras oncogenes leads to the abrogation of this arrest and to the p53-dependent apoptosis occurring in G1 phase of the cell cycle. Suppression of apoptosis by p53 gene knock-out, transduction of dominant negative p53 mutant or bcl-2 prevents death in suspension and greatly induces frequency of colony formation in semi-solid media. The ability of cells to undergo apoptosis does not correlate with their tumorigenicity in nude mice but does correlate with their ability to survive in lungs of intravenously injected mice and to form experimental metastases. We suggest that abrogation of a p53-mediated apoptosis facilitates experimental metastasis by promoting survival of tumor cells in circulation.

Animals↗