PubMed Health⌕ Search

Biomedical subjects

Andrei V Gudkov

Publications and source records attributed to Andrei V Gudkov.

36 records · Page 2Linked to original sources
PubMed2004

The ability of protein tyrosine phosphatase SHP-1 to suppress NFkappaB can be inhibited by dominant negative mutant of SIRPalpha.

In contrast with hematopoietic cells and fibroblasts, which express mainly one form of protein tyrosine phosphatase (PTP) SHP-1 or SHP-2, epithelial cells like A431, HeLa, and 293 express both forms of PTP. These two PTP regulate NFkappaB activity differently; SHP-1 inhibits and SHP-2 stimulates NFkappaB activation. In epithelial cells the process of NFkappaB activation depends on the combination of two PTP activities. The activity of PTP SHP-1 dominates in this tandem according to our data. The signal regulatory protein (SIRPalpha) is the adapter and the substrate of PTP SHP-1 and SHP-2. We investigated the role of SIRPalpha and its dominant negative mutant in PTP activities in 293 cells. The overexpression of wild-type SIRPalpha suppresses the activities of both PTP, but has a stronger effect on PTP SHP-2, especially when this protein is overexpressed in 293 cells. In contrast with wild-type SIRPalpha, its dominant negative mutant acts predominantly against PTP SHP-1, and can be detected in the complex with PTP SHP-1. The expression of dominant negative mutant of SIRPalpha has an effect similar to the expression of dominant negative PTP SHP-1 in the process of NFkappaB activation.

Antigens, Differentiation↗
PubMed2003

BMAL1-dependent circadian oscillation of nuclear CLOCK: posttranslational events induced by dimerization of transcriptional activators of the mammalian clock system.

Mammalian CLOCK and BMAL1 are two members of bHLH-PAS-containing family of transcription factors that represent the positive elements of circadian autoregulatory feedback loop. In the form of a heterodimer, they drive transcription from E-box enhancer elements in the promoters of responsive genes. We have examined abundance, posttranslational modifications, cellular localization of endogenous and ectopically expressed CLOCK and BMAL1 proteins. Nuclear/cytoplasm distribution of CLOCK was found to be under circadian regulation. Analysis of subcellular localization of CLOCK in embryo fibroblasts of mice carrying different germ-line circadian mutations showed that circadian regulation of nuclear accumulation of CLOCK is BMAL1-dependent. Formation of CLOCK/BMAL1 complex following ectopic coexpression of both proteins is followed by their codependent phosphorylation, which is tightly coupled to CLOCK nuclear translocation and degradation. This binding-dependent coregulation is specific for CLOCK/BMAL1 interaction, as no other PAS domain protein that can form a complex with either CLOCK or BMAL1 was able to induce similar effects. Importantly, all posttranslational events described in our study are coupled with active transactivation complex formation, which argues for their significant functional role. Altogether, these results provide evidence for an additional level of circadian system control, which is based on regulation of transcriptional activity or/and availability of CLOCK/BMAL1 complex.

3T3 Cells↗
PubMed2003

Melanoma cells can tolerate high levels of transcriptionally active endogenous p53 but are sensitive to retrovirus-transduced p53.

Malignant melanomas are frequently characterized by elevated levels of wild-type p53, suggesting that p53 function could be suppressed by a mechanism different from p53 mutation. We analysed the functionality of the p53-signaling pathway in a panel of seven human melanoma cell lines consisting of one p53-deficient line, two lines with mutant p53, and four lines expressing wild-type p53. Only lines with wild-type p53 were characterized by elevated levels of endogenous p21, high activity of p53-responsive reporters and accumulation of p53 in response to genotoxic stress, common properties of functional p53. The presence of wild-type p53 was associated with depletion or loss of p14ARF and p16 expression. The levels of p33ING1b and p24ING1c, two major products of Ing1 locus and putative coregulators of p53, were elevated in all cell lines tested; however, ectopic expression of either ING1 isoform had no effect on cell proliferation. All lines retained expression of Apaf-1, and all but one remained sensitive to ectopic expression of retrovirus-transduced p53. Our data indicate that regardless of abnormally high levels of p53 in melanomas, their p53 remains competent in transactivation of its targets, and, if highly overexpressed, capable of growth inhibition. Hence, the p53 pathway in malignant melanomas can be considered for pharmacological targeting and anticancer gene therapy.

Apoptotic Protease-Activating Factor 1↗
PubMed2003

Cooperation of two mutant p53 alleles contributes to Fas resistance of prostate carcinoma cells.

Both inactivation of p53 function and loss of sensitivity to Fas contribute to a malignant phenotype and frequently occur during tumor progression. Although in the majority of cases only one of the p53 alleles is mutated, some tumors acquire mutations in both alleles of the p53 gene. To determine the biological significance of this phenomenon, we analyzed p53 mutants, p53(223Leu) and p53(274Phe), from Fas-resistant prostate carcinoma cell line DU145. Both mutants differed from wild-type p53 in their conformation, transactivation ability, and effect on the growth of p53-deficient cells, with p53(223Leu) being more similar to wild-type p53 than was p53(274Phe). Interestingly, the biological effect of coexpression of the DU145-derived mutants was dramatically different from that of each mutant expressed alone. Whereas neither of the two mutants was found to be dominant-negative against wild-type p53, each neutralized the other's growth-suppressive effects and, in combination, were capable of down-regulating Fas expression and converting Fas-sensitive prostate carcinoma cells PC3 into Fas-resistant ones. These results indicate that two different p53 mutants that are separately rather weak can cooperate to generate p53 protein with anti-Fas function that is likely to provide additional selective advantages to the tumor.

Alleles↗
PubMed2003

p53 inhibitor pifithrin alpha can suppress heat shock and glucocorticoid signaling pathways.

Pifithrin alpha (PFTalpha) is a chemical compound isolated for its ability to suppress p53-mediated transactivation. It can protect cells from p53-mediated apoptosis induced by various stimuli and reduce sensitivity of mice to gamma radiation. Identification of molecular targets of PFTalpha is likely to provide new insights into mechanisms of regulation of p53 pathway and is important for predicting potential risks associated with administration of PFTalpha-like p53 inhibitors in vivo. We found that PFTalpha, in addition to p53, can suppress heat shock and glucocorticoid receptor signaling but has no effect on nuclear factor-kappaB signaling. PFTalpha reduces activation of heat shock transcription factor (HSF1) and increases cell sensitivity to heat. Moreover, it reduces activation of glucocorticoid receptor and rescues mouse thymocytes in vitro and in vivo from apoptotic death after dexamethasone treatment. PFTalpha affected both signaling pathways in a p53-independent manner. These observations suggest that PFTalpha targets some unknown factor that is common for three major signal transduction pathways.

Animals↗
PubMed2003

Paradoxical role of apoptosis in tumor progression.

Tumors frequently acquire resistance to apoptosis that is expected to contribute to malignant phenotype and reduce sensitivity to treatment. In fact, inactivation of p53 tumor suppressor gene resulting in suppression of apoptosis serves as a negative prognostic marker. Surprisingly, expression of a strong anti-apoptotic protein Bcl-2, another mechanism to avoid apoptosis, was found to be associated with a favorable prognosis. This paradoxical anti-progressor function of Bcl-2 has been explained in literature based on the negative effect of Bcl-2 on cell proliferation. Here, by analyzing accumulated experimental and clinical data, we provide evidence supporting another hypothesis that defines apoptosis as an accelerator of tumor progression. The mechanism of anti-progressor function of Bcl-2 is based on creation of tumors that maintain control of genomic stability by eliminating selective advantages for the cells that acquire resistance to apoptosis through loss of p53. Thus, inhibition of apoptosis does not lead to loss of genomic stability and creates tumor environment that no longer supports further tumor progression and inhibitors of apoptosis can be considered as factors suppressing tumor progression.

Animals↗
PubMed2003

Identification of new drug sensitivity genes using genetic suppressor elements: protein arginine N-methyltransferase mediates cell sensitivity to DNA-damaging agents.

Genetic suppressor elements (GSEs) are cDNA fragments encoding either truncated proteins, acting as dominant-negative mutants, or inhibitory antisense RNA segments counteracting with the gene from which they are derived. To identify genes controlling the cell response to cytotoxic agents, a normalized retroviral library of randomly fragmented cDNAs from Chinese hamster cell line DC-3F was screened for GSEs conferring resistance to the topoisomerase II inhibitor 9-OH-ellipticine. From 218 cDNA fragments isolated, 11 functional GSEs, corresponding to at least 8 independent genes, were selected. The gene corresponding to the most abundant GSE encodes two proteins, p77 and p82, highly homologous to proteins detected in various species and carrying the sequence motifs characteristic of the protein arginine N-methyltransferase family. Furthermore, a methylase activity was observed on myelin basic protein in immunoprecipitates of hemagglutinin-tagged p77 and p82. Therefore, p77 and p82 are the first identified members of a new protein arginine N-methyltransferase family. A decreased expression of these enzymes is associated with either resistance or hypersensitivity to a broad range of DNA-damaging agents. Our data indicate that down-regulation of these enzymes in the GSE-expressing cells would alter one or several steps downstream of the drug-target interaction in the drug-response pathway.

Amino Acid Sequence↗
PubMed2003

The role of p53 in determining sensitivity to radiotherapy.

Ionizing radiation (IR) has proven to be a powerful medical treatment in the fight against cancer. Rational and effective use of its killing power depends on understanding IR-mediated responses at the molecular, cellular and tissue levels. Tumour cells frequently acquire defects in the molecular regulatory mechanisms of the response to IR, which sensitizes them to radiation therapy. One of the key molecules involved in a cell's response to IR is p53. Understanding these mechanisms indicates new rational approaches to improving cancer treatment by IR.

Humans↗
PubMed2002

Dominant negative form of signal-regulatory protein-alpha (SIRPalpha /SHPS-1) inhibits tumor necrosis factor-mediated apoptosis by activation of NF-kappa B.

Genetic suppressor element (GSE) methodology was applied to identify new genes controlling cell response to tumor necrosis factor (TNF). A retroviral library of randomly fragmented normalized cDNA from mouse fibroblasts was screened for GSEs capable of protecting NIH3T3 cells from TNF-induced apoptosis. The most abundant among isolated GSEs represented a fragment of cDNA encoding the C-terminal cytoplasmic region of the immunoglobulin family inhibitory receptor, SHPS-1 (mouse homologue of human SIRPalpha). Ectopic expression of this fragment (both from human and mouse versions) increased the NF-kappaB-dependent transcription in three cell lines tested; this effect could be reduced by the expression of full-length SIRPalpha, suggesting that the isolated GSE acts through a dominant negative mechanism. GSE-mediated activation of NF-kappaB depended on the presence of serum, was abrogated by wortmannin, and was associated with phosphorylation of PKB/Akt, suggesting that Akt mediates it. These data indicate that SIRPalpha/SHPS-1 is involved in negative regulation of NF-kappaB signaling.

3T3 Cells↗
PubMed2002

Cdk4 disruption renders primary mouse cells resistant to oncogenic transformation, leading to Arf/p53-independent senescence.

A large number of human cancers display alterations in the Ink4a/cyclin D/Cdk4 genetic pathway, suggesting that activation of Cdk4 plays an important role in oncogenesis. Here we report that Cdk4-null mouse embryonic fibroblasts are resistant to transformation in response to Ras activation with dominant-negative (DN) p53 expression or in the Ink4a/Arf-null background, judged by foci formation, anchorage-independent growth, and tumorigenesis in athymic mice. Cdk4-null fibroblasts proliferate at normal rates during early passages. Whereas Cdk4(+/+)Ink4a/Arf(-/-) cells are immortal in culture, Cdk4(-/-)Ink4a/Arf(-/-) cells undergo senescence during continuous culture, as do wild-type cells. Activated Ras also induces premature senescence in Cdk4(-/-)Ink4a/Arf(-/-) cells and Cdk4(-/-) cells with DNp53 expression. Thus, Cdk4 deficiency causes senescence in a unique Arf/p53-independent manner, which accounts for the loss of transformation potential. Cdk4-null cells express high levels of p21(Cip1/Waf1) with increased protein stability. Suppression of p21(Cip1/Waf1) by small interfering RNA (siRNA), as well as expression of HPV-E7 oncoprotein, restores immortalization and Ras-mediated transformation in Cdk4(-/-)Ink4a/Arf(-/-) cells and Cdk4(-/-) cells with DNp53 expression. Therefore, Cdk4 is essential for immortalization, and suppression of Cdk4 could be a prospective strategy to recruit cells with inactive Arf/p53 pathway to senescence.

ADP-Ribosylation Factor 1↗
PubMed2002

Falkor, a novel cell growth regulator isolated by a functional genetic screen.

A novel cell growth regulator, named Falkor, was identified using a functional approach to mammalian gene cloning, the Genetic Supressor Elements (GSE) method. In this screen, expression of the C-terminal domain of Falkor conferred cells with resistance to cisplatin-induced growth arrest. Expression of the C-terminus of Falkor, but not of the full-length protein, enhanced cell growth both following genotoxic stress and under normal conditions suggesting a general role for this protein in cell growth control. This effect of the C-terminus fragment was abrogated by over-expression of the full-length Falkor, suggesting that the fragment counteracts the function of the full-length protein. Falkor is encoded by a 2-kb mRNA which is present at different levels in various tissues, and is localized in the nucleus of cells. The C-terminal domain of Falkor, isolated from the GSE library, has significant homology to a known human and rat cell growth regulator, SM-20, and to the C. elegans protein EGL-9, recently shown to modify the Hypoxia Inducible Factor-1alpha. The homology suggests that these proteins share a functional domain that is conserved among a family of growth regulation proteins.

Amino Acid Sequence↗
PubMed2002

Identification of a novel stress-responsive gene Hi95 involved in regulation of cell viability.

cDNA microarray hybridization was used in an attempt to identify novel genes participating in cellular responses to prolonged hypoxia. One of the identified novel genes, designated Hi95 shared significant homology to a p53-regulated GADD family member PA26. In addition to its induction in response to prolonged hypoxia, the increased Hi95 transcription was observed following DNA damage or oxidative stress, but not following hyperthermia or serum starvation. Whereas induction of Hi95 by prolonged hypoxia or by oxidative stress is most likely p53-independent, its induction in response to DNA damaging treatments (gamma- or UV-irradiation, or doxorubicin) occurs in a p53-dependent manner. Overexpression of Hi95 full-length cDNA was found toxic for many types of cultured cells directly leading either to their apoptotic death or to sensitization to serum starvation and DNA damaging treatments. Unexpectedly, conditional overexpression of the Hi95 cDNA in MCF7-tet-off cells resulted in their protection against cell death induced by hypoxia/glucose deprivation or H(2)O(2). Thus, Hi95 gene seems to be involved in complex regulation of cell viability in response to different stress conditions.

Amino Acid Sequence↗
PubMed2002

Bisindolylmaleimide IX facilitates tumor necrosis factor receptor family-mediated cell death and acts as an inhibitor of transcription.

Bisindolylmaleimides (Bis) were originally described as protein kinase C inhibitors. Several studies have shown that Bis potentiate tumor necrosis factor (TNF) receptor family-mediated apoptosis in lymphoid and dendritic cells, but the inhibition of protein kinase C cannot account for these effects (Zhou, T., Song, L., Yang, P., Wang, Z., Lui, D., and Jope, R. S. (1999) Nat. Med. 5, 42-48). We investigated the effect of four Bis derivatives (I, II, VIII, and IX) in human prostatic carcinoma cell lines and found that Bis IX was the most potent inducer of apoptosis under simultaneous treatment with TNF-alpha, agonistic anti-Fas monoclonal antibody, and TNF-related apoptosis-inducing ligand (TRAIL). Bis IX synergistically induced caspase activity in combination with apoptosis-inducing ligands and converted the phenotype of cell lines from apoptosis-resistant to -sensitive. Bis IX induced p53 accumulation in LNCaP (lymph node carcinoma of prostate), which expresses wild-type p53 that was not accompanied by the induction of p53-responsive genes, p21/WAF1, and Mdm2. Moreover, the induction of p21/WAF1 and Mdm2 by doxorubicin was abrogated by simultaneous treatment with Bis IX. These effects apparently result from general inhibition of transcription by Bis IX. We have shown by Northern blot analysis that the transcription activity of the hygromycin gene after transient transfection of pcDNA3.1-Hygro plasmid in 293 and HeLa cells was inhibited by Bis IX in a dose-dependent manner. Moreover, DNA binding activity of Bis IX was prevented by actinomycin D, suggesting that actinomycin D and Bis IX have similar mechanisms of interaction with DNA. In addition, we found that actinomycin D and Bis IX induced caspase activity to the same extent during TRAIL-mediated apoptosis. In summary, these results suggest that Bis IX potentiates TNF receptor family-mediated cell death in part as an inhibitor of transcription.

Apoptosis↗
PubMed2002

Expression of prostate specific antigen (PSA) is negatively regulated by p53.

Although prostate-specific antigen (PSA) is considered a uniquely important tumor marker and is broadly used for early detection of prostate cancer, the molecular mechanisms underlying its elevated expression in tumors have been unknown. By using cDNA microarray gene expression profiling, we found a fourfold increase in the PSA mRNA level in prostatic carcinoma cell line LNCaP, in which the p53 pathway was suppressed by a dominant negative p53 mutant. Consistently, p53 suppression caused a 4-8-fold increase in secretion of PSA protein in culture medium, suggesting that PSA gene expression is under negative control of p53. While wild type p53 strongly repressed, dominant negative p53 mutants stimulated PSA promoter-driven transcription and secretion of PSA in transient transfection experiments. The inhibitory effect of wild type p53 was undetectable in the presence of trichostatin A, suggesting the involvement of histone deacetylation in negative regulation of PSA promoter activity. Thus, PSA is likely to be a tissue specific indicator of transformation-associated p53 suppression in prostate cells. This finding provides a plausible explanation for a frequent increase of PSA levels in advanced prostate cancer.

Acetylation↗
PubMed2002

Unstable receptors disappear from cell surface during poliovirus infection.

BACKGROUND: Cellular receptors play a significant role in pathogenesis of viral infections. Previously, we demonstrated that TNFa receptor (TNFR1) rapidly disappeared from the cell surface upon poliovirus infection, whereas FAS was much more stable [1]. We suggested that the rate of decrease in receptor presentation on the surface of infected cells might reflect its turnover rate on uninfected cells. MATERIAL/METHODS: To test this hypothesis, we estimated by FACS analysis the turnover rates of receptors for TRAIL (TRAILR1 and TRAILR2), signal regulatory protein SIRPa, receptor for alpha/beta interferon (INFR1), and poliovirus receptor (CD155) on the surface of HeLa cells after the treatment with brefeldin A (to stop receptor replenishment through the Golgi-mediated trafficking) or poliovirus infection. RESULTS: A good correlation between turnover rates caused by the two interventions was observed, with the stability of receptor presentation changing in the following order: TRAILR1, TRAILR2, SIRPa (half-life on infected cells between 2-4 h) < INFR1 (4-6 h) < CD155 (>8 h, besides some early masking of the receptor by its binding of the virus). CONCLUSIONS: Our results suggest that disruption of the protein trafficking pathway during poliovirus infection leads to the diminished sensitivity of infected cells to pro-apoptotic factors, and thus represents one of the mechanisms by which virus modulates the host defense reactions.

Animals↗
PubMedDate not supplied

Apoptosis inhibitor as a suppressor of tumor progression: expression of Bcl-2 eliminates selective advantages for p53-deficient cells in the tumor.

Inactivation of p53 and expression of Bcl-2, frequently occurring during tumor progression, have different prognostic value: while inactivation of p53 is generally associated with unfavorable prognosis, expression of Bcl-2 often correlates with better clinical outcome and delays selection of metastatic variants of experimental tumors. To analyze the mechanisms underlying the "anti-progression" function of Bcl-2, we engineered tumor cell variants differing in their p53 status and Bcl-2 expression and compared their expansion in experimental tumors. Although neither p53 suppression nor Bcl-2-expression altered cell growth properties in vitro, both variants showed rapid accumulation in growing tumors in vivo, presumably due to their resistance to hypoxia. However, no expansion of p53-deficient variants occurred in the tumors formed by Bcl-2-overexpressing cells, indicating that p53 deficiency has no selective advantages in the Bcl-2-expressing environment. Importantly, expression of Bcl-2, unlike p53 suppression, did not lead to genomic instability as judged by the frequencies of gene amplification. Thus, acquisition of Bcl-2 expression is as advantageous for tumor cell growth in vivo as is p53 inactivation but does not affect genomic stability and creates the environment restrictive for the expansion of genetically unstable and potentially malignant p53-deficient cells, causing a delay in tumor progression and explaining the different prognostic value of Bcl-2 and p53.

Animals↗