Growth arrest induced by wild-type p53 protein blocks cells prior to or near the restriction point in late G1 phase.

Conditional expression of wild-type (wt) p53 protein in a glioblastoma tumor cell line has been shown to be growth inhibitory. We have now more precisely localized the position in the cell cycle where growth arrest occurs. We show that growth arrest occurs prior to or near the restriction point in late G1 phase of the cell cycle. The effect of wt p53 protein on the expression of four immediate-early genes (c-FOS, c-JUN, JUN-B, and c-MYC), one delayed-early gene (ornithine decarboxylase), and two late-G1/S-phase genes (B-MYB and DNA polymerase alpha) was also examined. Of this subset of growth response genes, only the expression of B-MYB and DNA polymerase alpha was significantly repressed. The possibility that decreased expression of B-MYB may be an important component of growth arrest mediated by wt p53 protein is discussed.     

Heterogeneity in therapeutic response of genetically altered myeloma cell lines to interleukin 6, dexamethasone, doxorubicin, and melphalan

Because there is no known genetic abnormality common to all patients with myeloma, it is important to understand how genetic heterogeneity may lead to differences in signal transduction, cell cycle, and response to therapy. Model cell lines have been used to study the effect that mutations in p53 and ras can have on growth properties and responses of myeloma cells. The U266 cell line has a single mutant p53 allele. Stable expression of wild-type (wt) p53 in U266 cells results in a significant suppression of interleukin (IL)-6 gene expression and in the concomitant suppression of cell growth that could be restored by the addition of exogenous IL-6. Expression of wt p53 also leads to cell cycle arrest and protection from doxorubicin (Dox)- and melphalan (Mel)-induced apoptosis. The addition of IL-6 resulted in cell cycle progression and blocked p53-mediated protection from apoptosis. ANBL6 is an IL-6-dependent cell line that is sensitive to dexamethasone (Dex), Dox, and Mel. IL-6 is able to protect ANBL6 cells from Dex- and Mel- but not Dox-induced apoptosis. To study the effect of an activating mutation in ras, the ANBL6 cell line transfected with either a constitutively activated N- or K-ras gene was used. Both N-ras12 and K-ras12 genes were able to protect ANBL6 cells from apoptosis induced by Dex, Dox, and Mel. These data show that changes in ras or p53 can alter the myeloma cell response to IL-6 and demonstrate that the genetic background can alter therapeutic responses.     

Dissection of the genetic programs of p53-mediated G1 growth arrest and apoptosis: blocking p53-induced apoptosis unmasks G1 arrest

Employing the myeloblastic leukemia M1 cell line, which does not express endogenous p53, and genetically engineered variants, it was recently shown that activation of p53, using a p53 temperature- sensitive mutant transgene (p53ts), resulted in rapid apoptosis that was delayed by high level ectopic expression of bcl-2. In this report, advantage has been taken of these M1 variants to investigate the relationship between p53-mediated G1 arrest and apoptosis. Flow cytometric cell cycle analysis has provided evidence that activation of wild-type (wt) p53 function in M1 cells resulted in the induction of G1 growth arrest; this was clearly seen in the M1p53/bcl-2 cells because of the delay in apoptosis that unmasked p53-induced G1 growth arrest. This finding was further corroborated at the molecular level by analysis of the expression and function of key cell cycle regulatory genes in M1p53 versus M1p53/bcl-2 cells after the activation of wt p53 function; events that take place at early times during the p53-induced G1 arrest occur in both the M1p53 and the M1p53/bcl-2 cells, whereas later events occur only in the M1p53/bcl-2 cells, which undergo delayed apoptosis, thereby allowing the cells to complete G1 arrest. Finally, it was observed that a spectrum of p53 target genes implicated in p53- induced growth suppression and apoptosis were similarly regulated, either induced (gadd45, waf1, mdm2, and bax) or suppressed (c-myc and bcl-2), after activation of wt p53 function in M1p53 and M1p53/bcl-2 cells. Taken together, these findings show that wt p53 can simultaneously induce the genetic programs of both G1 growth arrest and apoptosis within the same cell type, in which the genetic program of cell death can proceed in either G1-arrested (M1p53/bcl-2) or cycling (M1p53) cells. These findings increase our understanding of the functions of p53 as a tumor suppressor and how alterations in these functions could contribute to malignancy.     

转染p53基因对培养的人动脉平滑肌细胞的影响

目的:探讨转染野生型p53(wtp53)基因对培养的人动脉平滑肌细胞(HASMCs)的影响,为冠状动脉支架内再狭窄的治疗提供新的思路。方法:以Lipofectamine 2000脂质体介导wtp53基因转染HASMCs株后,用免疫组化染色法检测转染wtp53基因后,其编码蛋白在HASMCs中的表达。绘制wtp53基因转染后HASMCs的增殖曲线。用WST-1法测定A值,并计算细胞生长的抑制率。用流式细胞仪检测wtp53基因转染对HASMCs凋亡的影响。结果:免疫组化染色法显示,wtp53基因转染后,在HASMCs的细胞核内高表达;wtp53基因的表达可抑制HASMCs的体外生长。HASMC/wtp53细胞的生长曲线较对照组明显降低;WST-1法显示,HASMC/wtp53的细胞活力与对照组相比有显著性差异(P0.05)。流式细胞仪检测显示,转染wtp53基因的HASMCs的凋亡率达40%以上,显著高于对照组。结论:转染wtp53基因对HASMCs具有明显的抑制和凋亡作用,提示wtp53基因转染HASMCs技术有可能为人冠状动脉支架内再狭窄的治疗提供一种新的防治策略与手段。     

Inhibitory effect of tumor suppressor p33(ING1b) and its synergy with p53 gene in hepatocellular carcinoma

AIM: To investigate the inhibitory effect of tumor suppressor p33ING1b and its synergy with p53 gene in hepatocellular carcinoma (HCC). METHODS: Recombinant sense and antisense p33ING1b plasmids were transfected into hepatoma cell line HepG2 with lipofectamine. Apoptosis, G0/G1 arrest, cell growth rate and cloning efficiency in soft agar of HepG2 were analyzed after transfection. In three hepatoma cell lines with different endogenous p53 gene expressions, the synergistic effect of p33ING1b with p53 was analyzed by flow cytometry and luciferase assay was performed to detect the activation of p53 downstream gene p21WAF1/CIP1. In addition, the expression and mutation rates of p33ING1b in HCC tissues were measured by immunohistochemistry and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). RESULTS: Overexpression of p33ING1b inhibited cell growth of HepG2, induced more apoptosis and protected cells from growth in soft agar. Combined transfer of p33ING1b and p53 gene promoted hepatoma cell apoptosis, G0/G1 arrest and elevated expression of p21WAF1/CIP1. Immunostaining results showed co-localized P33ING1b with P53 protein in HCC tissues and there was a significant relation between protein expression rates of these two genes (P<0.01). Among 28 HCC samples, p33ING1b presented a low gene mutation rate (7.1%). CONCLUSION: p33ING1b collaborates with p53 in cell growth inhibition, cell cycle arrest and apoptosis in HCC. Loss or inactivation of p33ING1b normal function may be an important mechanism for the development of HCC retaining wildtype p53.     

Constitutive expression of B-myb can bypass p53-induced Waf1/Cip1-mediated G1 arrest.

Overexpression of wild-type p53 protein has been shown to induce arrest in the G1 stage of the cell cycle and to transactivate expression of the gene that encodes the 21-kDa Waf1/Cip1 protein, a potent inhibitor of cyclin-dependent kinase activity. p53-dependent G1 arrest is accompanied by decreased expression of the B-myb gene, a relative of the c-myb cellular oncogene. In this study we show that B-myb expression is required for cells to progress from G1 into S phase and that high levels of ectopic B-myb expression uncoupled from cell cycle regulation rescues cells from p53-induced G1 arrest even in the presence of Waf1/Cip1 transactivation and inhibition of cyclin E/Cdk2 kinase activity. Cotransfection experiments with p53 expression plasmids and expression plasmids encoding in-frame deletion mutations in B-myb coding sequences indicate that the DNA-binding domain of the B-Myb protein is required for this activity. These results provide evidence of a bypass of p53-induced Waf1/Cip1-mediated cell cycle regulatory pathways by a member of the myb oncogene family.     

Wild-type p53 is a cell cycle checkpoint determinant following irradiation.

Cell cycle checkpoints appear to contribute to an increase in cell survival and a decrease in abnormal heritable genetic changes following exposure to DNA damaging agents. Though several radiation-sensitive yeast mutants have been identified, little is known about the genes that control these responses in mammalian cells. Recent studies from our laboratory have demonstrated a close correlation between expression of wild-type p53 genes in human hematopoietic cells and their ability to arrest in G1 phase after certain types of DNA damage. In the present study, this correlation was first generalized to nonhematopoietic mammalian cells as well. A cause and effect relationship between expression of wild-type p53 and the G1 arrest that occurs after gamma irradiation was then established by demonstrating (i) acquisition of the G1 arrest after gamma irradiation following transfection of wild-type p53 genes into cells lacking endogenous p53 genes and (ii) loss of the G1 arrest after irradiation following transfection of mutant p53 genes into cells with wild-type endogenous p53 genes. A defined role for p53 (the most commonly mutated gene in human cancers) in a physiologic pathway has, to our knowledge, not been reported previously. Furthermore, these experiments illustrate one way in which a mutant p53 gene product can function in a "dominant negative" manner. Participation of p53 in this pathway suggests a mechanism for the contribution of abnormalities in p53 to tumorigenesis and genetic instability and provides a useful model for studies of the molecular mechanisms of p53 involvement in controlling the cell cycle.     

Regulation of transcription by cyclic AMP-dependent protein kinase

cAMP-dependent protein kinase (PKA; ATP: protein phosphotransferase; EC 2.7.1.37) appears to be the major mediator of cAMP responses in mammalian cells. We have investigated the role of PKA subunits in the regulation of specific genes in response to cAMP by cotransfection of wild-type or mutant subunits of PKA together with cAMP-inducible reporter genes. Overexpression of catalytic subunit induced expression from three cAMP-regulated promoters (alpha-subunit, c-fos, E1A) in the absence of elevated levels of cAMP but did not affect expression from two unregulated promoters (Rous sarcoma virus, simian virus 40). Cotransfection of a regulatory subunit gene containing mutations in both cAMP binding sites strongly repressed both basal and induced expression from the cAMP-responsive alpha-subunit promoter without affecting expression from the Rous sarcoma virus promoter. These experiments indicate that cAMP induces gene expression through phosphorylation by the catalytic subunit and that the ambient degree of phosphorylation dictates the level of basal as well as induced expression of the cAMP-regulated alpha-subunit gene.     

Immunohistochemical analysis of p53, cyclinD1, RB1, c-fos and N-ras gene expression in hepatocellular carcinoma in Iran

AIM： TO study the effect of some genes especially those involved in cell cycle regulation on hepatocellular carcinoma.
METHODS： Paraffin-embedded tissue samples of 25 patients （18 males and 7 females） with hepatocellular carcinoma were collected from 22 pathology centers in Tehran during 2000-2001, and stained using immunohistochemistry method （avidin-biotin-peroxidase） for detection of p53, cyclinD1, RB1, c-los and N-ras proteins. RESULTS： Six （24%）, 5 （20%）, 12 （48%） and 2 samples （8%） were positive for p53, cyclinDl, C-los and N-ras expression, respectively. Twenty-two （88%） samples had alterations in the （31 cell-cycle checkpoint protein expression （RBI or cyclinD1）. P53 positive samples showed a higher （9 times） risk of being positive for RBI protein than p53 negative samples. Loss of expression of RBI in association with p53 over-expression was observed in 4 （66.7%） of 6 samples. Loss of expression of RBI was seen in all cyclinD1 positive, 20 （90.9%） N-ras negative, and ii （50%） C-fos positive samples, respectively. CyclinD1 positive samples showed a higher （2.85 and 4.75 times） risk of being positive for c-los and N-ras expression than cyclinD1 negative samples.
CONCLUSION： The expression of p53, RB1 and c-los genes appears to have a key role in the pathogenesis of hepatocellular carcinoma in Iran. Simultaneous overexpression of these genes is significantly associated with their loss of expression during development of hepatocellular carcinoma.     

Cooperative action of germ-line mutations in decorin and p53 accelerates lymphoma tumorigenesis

Ectopic expression of decorin in a wide variety of transformed cells results in growth arrest and the inability to generate tumors in nude mice. This process is caused by a decorin-mediated activation of the epidermal growth factor receptor, which leads to a sustained induction of endogenous p21(WAF1/CIP1) (the cyclin-dependent kinase inhibitor p21) and growth arrest. However, mice harboring a targeted disruption of the decorin gene do not develop spontaneous tumors. To test the role of decorin in tumorigenesis, we generated mice lacking both decorin and p53, an established tumor-suppressor gene. Mice lacking both genes showed a faster rate of tumor development and succumbed almost uniformly to thymic lymphomas within 6 months [mean survival age (T50) approximately 4 months]. Mice harboring one decorin allele and no p53 gene developed the same spectrum of tumors as the double knockout animals, but had a survival rate similar to the p53 null animals (T50 approximately 6 months). Ectopic expression of decorin in thymic lymphoma cells isolated from double mutant animals markedly suppressed their colony-forming ability. When these lymphoma cells were cocultured with fibroblasts derived from either wild-type or decorin null embryos, the cells grew faster in the absence of decorin. Moreover, exogenous decorin proteoglycan or its protein core significantly retarded their growth in vitro. These results indicate that the lack of decorin is permissive for lymphoma tumorigenesis in a mouse model predisposed to cancer and suggest that germ-line mutations in decorin and p53 may cooperate in the transformation of lymphocytes and ultimately lead to a more aggressive phenotype by shortening the tumor latency.