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Biomedical subjects

J A Pietenpol

Publications and source records attributed to J A Pietenpol.

At least 19 recordsLinked to original sources
PubMed2002

Cell cycle checkpoint signaling: cell cycle arrest versus apoptosis.

Although toxicants may initiate cell damage or stress, the cellular proteins that are involved in control of cell cycle and apoptosis are the final arbiters of cell fate. The biochemical pathways that restrain cell cycle transition and/or induce cell death after stress are known as cell cycle checkpoints. These checkpoints maintain the fidelity of DNA replication, repair, and division. Herein, select cell cycle checkpoint signaling pathways will be discussed and how different components of these pathways are regulated by exogenous and endogenous agents, with focus on the p53 tumor suppressor signaling. The p53 protein is known to play a key role in growth arrest and apoptosis after cell stress, primarily through its ability to regulate the transcription of select downstream target genes in the cell. Further elucidation of the signaling pathways that control growth arrest and apoptosis will continue to provide insights to the complex cellular responses to environmental toxicants.

Animals↗
PubMed2001

A-204197, a new tubulin-binding agent with antimitotic activity in tumor cell lines resistant to known microtubule inhibitors.

Drug resistance is a prevalent problem in the treatment of neoplastic disease, and the effectiveness of many clinically useful drugs is limited by the fact that they are substrates for the efflux pump, P-glycoprotein. Because there is a need for new compounds that are effective in treating drug-resistant tumors, we tested A-204197 (4-[4-acetyl-4,5-dihydro-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]-N,N-dimethylbenzeneamine), a novel oxadiazoline derivative with antiproliferative properties, on cell lines that were either sensitive or resistant to known microtubule inhibitors. Cell lines that were resistant to paclitaxel, vinblastine, or colchicine were equally sensitive to A-204197 (proliferation IC50s ranging from 36 to 48 nM) despite their expression levels of P-glycoprotein. The effect of A-204197 on cell growth was associated with cell cycle arrest in G2-M, increased phosphorylation of select G2-M checkpoint proteins, and apoptosis. In competition-binding assays, A-204197 competed with [3H]-labeled colchicine for binding to tubulin (K(i) = 0.75 microM); however, it did not compete with [3H]-labeled paclitaxel. A-204197 prevented tubulin polymerization in a dose-dependent manner (IC50 = 4.5 microM) in vitro and depolymerized microtubules in a time-dependent manner in cultured cells. These findings indicate A-204197 is a promising new tubulin-binding compound with antimitotic activity that has potential for treating neoplastic diseases with greater efficacy than currently used antimitotic agents.

Antineoplastic Agents↗
PubMed2001

Increased p53 phosphorylation after microtubule disruption is mediated in a microtubule inhibitor- and cell-specific manner.

p53 is present at low levels in unstressed cells. Numerous cellular insults, including DNA damage and microtubule disruption, elevate p53 protein levels. Phosphorylation of p53 is proposed to be important for p53 stabilization and activation after genotoxic stress; however, p53 phosphorylation after microtubule disruption has not been analysed. The goal of the current study was to determine if p53 phosphorylation increases after microtubule disruption, and if so, to identify specific p53 residues necessary for microtubule inhibitor-induced phosphorylation. Two dimensional gel analyses demonstrated that the number of p53 phospho-forms in cells increased after treatment with microtubule inhibitors (MTIs) and that the pattern of p53 phosphorylation was distinct from that observed after DNA damage. p53 phosphorylation also varied in a MTI-dependent manner, as Taxol and Vincristine induced more p53 phospho-forms than nocodazole. Further, MTI treatment increased phosphorylation of p53 on serine-15 in epithelial tumor cells. In contrast, serine-15 phosphorylation of p53 did not increase in MTI-treated primary cultures of human fibroblasts. Analysis of ectopically expressed p53 phospho-mutant proteins from Taxol- and nocodazole-treated cells indicated that multiple p53 amino terminal residues, including serine-15 and threonine-18, were required for Taxol-mediated phosphorylation of p53. Taken together, the results of this study demonstrate that distinct p53 phospho-forms are induced by MTI treatment as compared to DNA damage and that p53 phosphorylation is mediated in a MTI- and cell-specific manner. Oncogene (2001) 20, 113 - 124.

Antineoplastic Agents↗
PubMed2001

4-hydroxynonenal induces apoptosis via caspase-3 activation and cytochrome c release.

We investigated the mechanism by which 4-hydroxynonenal (HNE), a major aldehydic product of lipid peroxidation, induces apoptosis in tumor cells. Treatment of human colorectal carcinoma (RKO) cells with HNE-induced poly-ADP-ribose-polymerase (PARP) cleavage and DNA fragmentation in a dose- and time-dependent manner. The induction of PARP cleavage and DNA fragmentation paralleled caspase-2, -3, -8, and -9 activation. Pretreatment of cells with an inhibitor of caspase-3, z-DEVD-fmk, or a broad spectrum caspase inhibitor, z-VAD-fmk, abolished caspase activation and subsequent PARP cleavage. Constitutive expression of high levels of Bcl-2 protected cells from HNE-mediated apoptosis. In addition, Bcl-2 overexpression inhibited cytochrome c release from mitochondria and subsequent caspase-2, -3, and -9 activation. These findings demonstrate that HNE triggers apoptotic cell death through a mitochondrion-dependent pathway involving cytochrome c release and caspase activation. Bcl-2 overexpression protected cells from HNE-induced apoptosis through inhibition of cytochrome c release.

Aldehydes↗
PubMed2001

Kinetics of p53 binding to promoter sites in vivo.

Downstream target genes of p53 are thought to mediate its tumor-suppressive activity, but it is unknown whether differential transactivation of these genes is regulated at the level of p53 binding to their promoters. To address this issue, p53 binding in vivo to consensus sites in the p21(Waf1), MDM2, and PIG3 promoters was investigated in cells exposed to adriamycin (ADR) or ionizing radiation as well as in an inducible p53 cell line. p53-DNA complexes were cross-linked in vivo by treating the cells with formaldehyde and processed by chromatin immunoprecipitation-PCR. This methodology allowed for the analysis of relevant p53-DNA complexes by preventing redistribution of cellular components upon collection of cell extracts. Increased p53 binding to the p21(Waf1), MDM2, and PIG3 promoters occurred within 2 h after p53 activation; however, significant increases in PIG3 transcription did not occur until 15 h after p53 binding. Gel shift analyses indicated that p53 had lower affinity for the consensus binding site in the PIG3 promoters compared to its consensus sites in the p21 and MDM2 genes, which suggests that additional factors may be required to stabilize the interaction of p53 with the PIG3 promoter. Further, acetylated p53 (Lys382) was found in chemically cross-linked complexes at all promoter sites examined after treatment of cells with ADR. In summary, the kinetics of p53 binding in vivo to target gene regulatory regions does not uniformly correlate with target gene mRNA expression for the p53 target genes examined. Our results suggest that target genes with low-affinity p53 binding sites may require additional events and will have delayed kinetics of induction compared to those with high-affinity binding sites.

Gene Expression Regulation, Neoplastic↗
PubMed2000

Syk: a new player in the field of breast cancer.

Breast tumor development and progression are thought to occur through a complex, multistep process, including oncogene activation (eg HER2/neu) and mutation or loss of tumor suppressor genes (eg p53). Determining the function of genetic alterations in breast carcinoma tumorigenesis and metastasis has been the focus of intensive research efforts for several decades. One group of proteins that play a critical role in breast cancer cell signaling pathways are tyrosine kinases. Overexpression of the tyrosine kinase HER2/neu is observed in many human breast cancers and is positively correlated with enhanced tumorigenesis. Recently, another tyrosine kinase, Syk, has been implicated as an important inhibitor of breast cancer cell growth and metastasis. This recent finding was unexpected, since Syk function has been predominantly linked to hematopoietic cell signaling, and is discussed further in this commentary.

Animals↗
PubMed2000

p53-dependent expression of PIG3 during proliferation, genotoxic stress, and reversible growth arrest.

The p53-inducible gene 3 (PIG3) was recently identified in a screen for genes induced by p53 before the onset of apoptosis. PIG3 shares significant homology with oxidoreductases from several species. In this study, PIG3-specific antibodies were used to analyze cellular PIG3 protein levels under control and genotoxic stress conditions. PIG3 protein was localized to the cytoplasm and induced in primary, non-transformed, and transformed cell cultures after exposure to genotoxic agents. The induction of PIG3 was p53-dependent and occurred with delayed kinetics as compared with other p53 downstream targets, such as p21 and MDM2. Using a p53-inducible cell model system, in which p53-mediated growth arrest is reversible, we found that PIG3 levels were increased during p53-mediated growth arrest. Interestingly, elevated levels of PIG3 were maintained in cells that resumed cycling in the absence of ectopic p53 expression, suggesting that PIG3 is a long-lived reporter, which may be useful for detecting transient activation of p53.

Apoptosis↗
PubMed2000

p53 regulation of G(2) checkpoint is retinoblastoma protein dependent.

In the present study, we investigated the role of p53 in G(2) checkpoint function by determining the mechanism by which p53 prevents premature exit from G(2) arrest after genotoxic stress. Using three cell model systems, each isogenic, we showed that either ectopic or endogenous p53 sustained a G(2) arrest activated by ionizing radiation or adriamycin. The mechanism was p21 and retinoblastoma protein (pRB) dependent and involved an initial inhibition of cyclin B1-Cdc2 activity and a secondary decrease in cyclin B1 and Cdc2 levels. Abrogation of p21 or pRB function in cells containing wild-type p53 blocked the down-regulation of cyclin B1 and Cdc2 expression and led to an accelerated exit from G(2) after genotoxic stress. Thus, similar to what occurs in p21 and p53 deficiency, pRB loss can uncouple S phase and mitosis after genotoxic stress in tumor cells. These results indicate that similar molecular mechanisms are required for p53 regulation of G(1) and G(2) checkpoints.

G2 Phase↗
PubMed1999

Helicobacter pylori strain-specific genotypes and modulation of the gastric epithelial cell cycle.

Helicobacter pylori cag+ strains enhance gastric epithelial cell proliferation and attenuate apoptosis in vivo, which may partially explain the increased risk of gastric cancer associated with these strains. The goals of this study were to identify specific H. pylori genes that regulate epithelial cell cycle events and determine whether these effects were dependent upon p53-mediated pathways. AGS gastric epithelial cells were cultured alone or in the presence of 21 clinical H. pylori isolates, H. pylori reference strain 60190, or its isogenic cagA-, picB-, vacA-, or picB-/vacA- derivatives. Coculture of H. pylori with AGS cells significantly decreased cell viability, an effect most prominent with cag+ strains (P < 0.001 versus cag-strains). cag+ strains significantly increased progression of AGS cells from G1 into G2-M at 6 h and enhanced apoptosis by 72 h. Compared with the parental 60190 strain, the picB- mutant attenuated cell cycle progression at 6 h (P < or = 0.05), and decreased apoptosis with enhanced AGS cell viability at 24 h (P < or = 0.04). The vacA- mutant decreased apoptosis and enhanced viability at later (48-72 h) time points (P < or = 0.05). Compared with the wild-type strain, the picB-/vacA- double mutant markedly attenuated apoptosis and increased cell viability at all time points (P < or = 0.05). Furthermore, cocolonization with H. pylori had no significant effect on expression of p53, p21, and MDM2. The diminished AGS cell viability, progression to G2-M, and apoptosis associated with cag+ H. pylori strains were dependent upon expression of vacA and genes within the cag pathogenicity island. These results may explain heterogeneity in levels of gastric epithelial cell proliferation and apoptosis found within H. pyloricolonized mucosa.

Apoptosis↗
PubMed1999

Defective G1-S cell cycle checkpoint function sensitizes cells to microtubule inhibitor-induced apoptosis.

Defective cell cycle checkpoint function has been linked to enhanced sensitivity of tumor cells to certain genotoxic agents. To determine whether loss of the G1-S checkpoint function would sensitize tumor cells to microtubule inhibitor (MTI)-induced apoptosis, we examined the effect of the MTIs, Taxol and vincristine, on the cell cycle kinetics and survival of two isogenic cell lines, HCT116 p21+/+ and HCT116 p21-/-, which differ only at the p21 locus. p21-deficient cells displayed a dose-dependent, enhanced chemosensitivity to MTIs in both monolayer and soft agar assays as well as in mice xenograft tumors. The increased sensitivity of the p21-deficient cells to MTIs correlated with prolonged cyclin B1/Cdc2 activity and the occurrence of endoreduplication. Furthermore, sensitivity of p53-deficient cells to MTI-induced apoptosis was significantly reduced by induction of ectopic p21 protein. The results suggest that the status of G1-S checkpoint function in tumor cells may be an important determinant in the efficacy of MTIs used clinically.

Animals↗
PubMed1999

High affinity insertion/deletion lesion binding by p53. Evidence for a role of the p53 central domain.

In addition to binding DNA in a sequence-specific manner, p53 can interact with nucleic acids in a sequence-independent manner. p53 can bind short single-stranded DNA and double-stranded DNA containing nucleotide loops; these diverse associations may be critical for p53 signal transduction. In this study, we analyzed p53 binding to DNA fragments containing insertion/deletion mismatches (IDLs). p53 required an intact central domain and dimerization domain for high affinity complex formation with IDLs. In fact, the C terminus of p53 (amino acids 293-393) was functionally replaceable with a foreign dimerization domain in IDL binding assays. From saturation binding studies we determined that the KD of p53 binding to IDLs was 45 pM as compared with a KD of 31 pM for p53 binding to DNA fragments containing a consensus binding site. Consistent with these dissociation constants, p53-IDL complexes were dissociated with relatively low concentrations of competitor consensus site-containing DNA. Although p53 has a higher affinity for DNA with a consensus site as compared with IDLs, the relative number and availability of each form of DNA in a cell immediately after DNA damage may promote p53 interaction with DNA lesions. Understanding how the sequence-specific and nonspecific DNA binding activities of p53 are integrated will contribute to our knowledge of how signaling cascades are initiated after DNA damage.

Animals↗
PubMed1999

Cell cycle checkpoints as therapeutic targets.

Most human breast tumors arise from multiple genetic changes which gradually transform differentiated and growth-limited cells into highly invasive cells that are unresponsive to growth controls. The genetic evolution of normal breast cells into cancer cells is largely determined by the fidelity of DNA replication, repair, and division. Cell cycle arrest in response to DNA damage is an important part of the mechanism used to maintain genomic integrity. The control mechanisms that restrain cell cycle transition after DNA damage are known as cell cycle checkpoints. This review will focus on cell cycle checkpoint signaling pathways commonly mutated in human breast tumors and suggest how different components of these checkpoint pathways offer the potential for chemotherapeutic intervention.

Animals↗
PubMed1999

p21(Waf1/Cip1) inhibition of cyclin E/Cdk2 activity prevents endoreduplication after mitotic spindle disruption.

During a normal cell cycle, entry into S phase is dependent on completion of mitosis and subsequent activation of cyclin-dependent kinases (Cdks) in G1. These events are monitored by checkpoint pathways. Recent studies and data presented herein show that after treatment with microtubule inhibitors (MTIs), cells deficient in the Cdk inhibitor p21(Waf1/Cip1) enter S phase with a >/=4N DNA content, a process known as endoreduplication, which results in polyploidy. To determine how p21 prevents MTI-induced endoreduplication, the G1/S and G2/M checkpoint pathways were examined in two isogenic cell systems: HCT116 p21(+/+) and p21(-/-) cells and H1299 cells containing an inducible p21 expression vector (HIp21). Both HCT116 p21(-/-) cells and noninduced HIp21 cells endoreduplicated after MTI treatment. Analysis of G1-phase Cdk activities demonstrated that the induction of p21 inhibited endoreduplication through direct cyclin E/Cdk2 regulation. The kinetics of p21 inhibition of cyclin E/Cdk2 activity and binding to proliferating-cell nuclear antigen in HCT116 p21(+/+) cells paralleled the onset of endoreduplication in HCT116 p21(-/-) cells. In contrast, loss of p21 did not lead to deregulated cyclin D1-dependent kinase activities, nor did p21 directly regulate cyclin B1/Cdc2 activity. Furthermore, we show that MTI-induced endoreduplication in p53-deficient HIp21 cells was due to levels of p21 protein below a threshold required for negative regulation of cyclin E/Cdk2, since ectopic expression of p21 restored cyclin E/Cdk2 regulation and prevented endoreduplication. Based on these findings, we propose that p21 plays an integral role in the checkpoint pathways that restrain normal cells from entering S phase after aberrant mitotic exit due to defects in microtubule dynamics.

CDC2-CDC28 Kinases↗
PubMed1999

Treatment with farnesyl-protein transferase inhibitor induces regression of mammary tumors in transforming growth factor (TGF) alpha and TGF alpha/neu transgenic mice by inhibition of mitogenic activity and induction of apoptosis.

Mouse mammary tumor virus-transforming growth factor alpha (MMTV-TGF alpha) and MMTV-TGF alpha/neu transgenic mice develop mammary tumors after a long latency and therefore provide useful model systems for breast cancer with its recognized activation of receptor tyrosine kinase signaling. We used these mice to study the antitumor effect of L-744,832 (FTI), a potent and selective inhibitor of farnesyl-protein transferase, and hence of Ras function. A total of 55 mice were assigned randomly to treatment with FTI or vehicle, and one-half of the mice were crossed over after initial treatment to the opposite group. L-744,832 induced reversible regression of mammary tumors that was paralleled by a decrease in serum levels of TGF alpha secreted by the tumor cells. There was no difference in response to treatment with FTI between MMTV-TGF alpha mice, in which tumorigenesis was accelerated by multiparity or the chemical carcinogen 7,12-dimethylbenzanthracene, and MMTV-TGF alpha/neu mice. The tumor histological type had no impact on FTI sensitivity. For mechanistic analyses, tumor excision biopsies were obtained from 12 mice before and after treatment with L-744,832. In these samples, tumor regression was paralleled biochemically by inhibition of mitogen-activated protein kinase activity and biologically by an increase in G1-phase and decrease in S-phase fractions, as well as induction of apoptosis. These results suggest that the potential clinical use of FTI could be expanded to include cancers harboring activated receptor tyrosine kinases as well as those containing activated Ras.

Alkyl and Aryl Transferases↗
PubMed1998

Mitotic phosphorylation of Bcl-2 during normal cell cycle progression and Taxol-induced growth arrest.

There is increasing evidence that prolonged mitotic arrest initiates apoptosis; however, little is known about the signaling pathways involved. Several studies have associated deregulated Cdc2 activity with apoptosis. Herein, we report that the anti-apoptotic protein, Bcl-2, undergoes cell cycle-dependent phosphorylation during mitosis when there is elevated Cdc2 activity. We found that paclitaxel (Taxol(R)) treatment of epithelial tumor cells induced a prolonged mitotic arrest, elevated levels of mitotic kinase activity, hyperphosphorylation of Bcl-2, and subsequent cell death. The Taxol-induced Bcl-2 phosphorylation was dose-dependent. Furthermore, phosphorylated Bcl-2 remained complexed with Bax in Taxol-treated cells undergoing apoptosis. Immunoprecipitation experiments revealed a Bcl-2-associated kinase capable of phosphorylating histone H1 in vitro. However, the kinase was likely not cyclin B1/Cdc2, since cyclin B1/Cdc2 was not detectable in Bcl-2 immunoprecipitates, nor was recombinant Bcl-2 phosphorylated in vitro by cyclin B1/Cdc2. The results of this study further define a link between mitotic kinase activation and the apoptotic machinery in the cell. However, the role, if any, of prolonged Bcl-2 phosphorylation in Taxol-mediated apoptosis awaits further definition of Bcl-2 mechanism of action. Taxol may increase cellular susceptibility to apoptosis by amplifying the normal downstream events associated with mitotic kinase activation.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Negative regulation of Wee1 expression and Cdc2 phosphorylation during p53-mediated growth arrest and apoptosis.

The G2 cell cycle checkpoint protects cells from potentially lethal mitotic entry after DNA damage. This checkpoint involves inhibitory phosphorylation of Cdc2 at the tyrosine-15 (Y15) position, mediated in part by the Wee1 protein kinase. Recent evidence suggests that p53 may accelerate mitotic entry after DNA damage and that the override of the G2 checkpoint may play a role in the induction of apoptosis by p53. To determine the biochemical mechanism by which p53 inactivates the G2 checkpoint, the effects of p53 activation on Wee1 expression, Cdc2-Y15 phosphorylation, and cyclin B1-associated Cdc2 kinase activity were examined. Under conditions of either growth arrest or apoptosis, p53 activation resulted in the down-regulation of Wee1 expression and dephosphorylation of Cdc2. A parallel increase in cyclin B1/Cdc2 kinase activity was observed during p53-mediated apoptosis. Negative regulation of the Wee1 expression and Cdc2 phosphorylation by p53 was also evident in thymus tissue from p53+/+ mice but not from p53-/- mice. Inactivation of the G2 checkpoint may contribute to the tumor suppressor activity of p53.

Animals↗
PubMed1998

The role of transforming growth factor beta in glioma progression.

This review examines the apparently paradoxical conversion of transforming growth factor beta's (TGFbeta) regulatory role as a growth inhibitor among normal glial cells to that of a progression factor among glioblastomas (GM). In vitro, TGFbeta functions as an autocrine growth inhibitor of near-diploid gliomas of any grade. In contrast, hyperdiploid glioblastoma multiforme (HD-GM) cultures proliferate in response to TGFbeta, which is mediated by induction of platelet-derived growth factor B chain (PDGF-BB). The dominant hypothesis of TGFbeta's pathogenetic association with malignant transformation has been predicated upon acquisition of resistance to its growth inhibitory effects. However, the lack of obvious correlation with TGFbeta receptor (TbetaR) expression (or loss) between the HD-GM and the TGFbeta-inhibited GM cultures suggests the existence of intrinsically opposed regulatory mechanisms influenced by TGFbeta. The mechanism of conversion might be explained either by the loss of a putative tumor suppressor gene (TSG) which mediates TGFbeta's inhibition of growth or by enhancement of an active oncogenic pathway among the HD-GM. The frequency of mutations within glioma-associated TSG, such as TP53 and RB, suggests that defects in TGFbeta's inhibitory signaling pathway may have analogous effects in the progression to HD-GM, and TGFbeta's conversion to a mitogen. Alternative sites of inactivation which might explain the loss of TGFbeta's inhibitory effect include inactivating mutation/loss of the TbetaR type II, alterations in post-receptor signal transmission or the cyclin/cyclin dependent kinase system which regulates the phosphorylation of pRB. Loss or inactivation of a glial TSG with a consequent failure of inhibition appears to allow TGFbeta's other constitutive effects, such as induction of c-sis, to become functionally dominant. Mechanistically, TGFbeta's conversion from autocrine inhibitor to mitogen promotes 'clonal dominance' by conferring a Darwinian advantage to the hyperdiploid subpopulations through qualitative and quantitative differences in its modulation of PDGF-A and c-sis, with concomitant paracrine inhibition of competing, near-diploid elements.

Animals↗