Effects of exogenous wild-type p53 on a human lung carcinoma cell line with endogenous wild-type p53

Several studies have shown that expression of exogenous wild-type p53 is detrimental to the growth of cell lines with absent or mutant p53. In this study, wild-type p53 cDNA expression plasmids were transfected into A549 lung carcinoma cells which had previously been shown by sequencing to contain wild-type p53. When a constitutively expressed wild-type p53 plasmid containing the neomycin resistance gene was transfected into these cells, no G418-resistant colonies contained the exogenous p53 cDNA even though the neomycin resistance gene was integrated. When cells were transfected with a dexamethasone-inducible wild-type p53 cDNA expression plasmid, induction of p53 expression resulted in a decreased growth rate and a decreased proportion of S-phase cells. Continuous treatment with dexamethasone resulted in continued p53 expression for 16 days, but beyond that time expression ceased and could not be reinduced. These data indicated that although the A549 cell line could proliferate in the presence of endogenous wild-type p53 there was a strong selection pressure against continued expression of additional exogenous wild-type p53.     

Mutant p53 tumor suppressor alleles release ras-induced cell cycle growth arrest.

Overexpression of an activated ras gene in the rat embryo fibroblast line REF52 results in growth arrest at either the G1/S or G2/M boundary of the cell cycle. Both the DNA tumor virus proteins simian virus 40 large T antigen and adenovirus 5 E1a are able to rescue ras induced lethality and cooperate with ras to fully transform REF52 cells. In this report, we present evidence that the wild-type activity of the tumor suppressor gene p53 is involved in the negative growth regulation of this model system. p53 genes encoding either a p53Val-135 or p53Pro-193 mutation express a highly stable p53 protein with a conformation-dependent loss of wild-type activity and the ability to eliminate any endogenous wild-type p53 activity in a dominant negative manner. In cotransfection assays, these mutant p53 genes are able to rescue REF52 cells from ras-induced growth arrest, resulting in established cell lines which express elevated levels of the ras oncoprotein and show morphological transformation. Full transformation, as assayed by tumor formation in nude mice, is found only in the p53Pro-193-plus-ras transfectants. These cells express higher levels of the ras protein than do the p53Val-135-plus-ras-transfected cells. Transfection of REF52 cells with ras alone or a full-length genomic wild-type p53 plus ras results in growth arrest and lethality. Therefore, the selective event for p53 inactivation or loss during tumor progression may be to overcome a cell cycle restriction induced by oncogene overexpression (ras). These results suggest that a normal function of p53 may be to mediate negative growth regulation in response to ras or other proliferative inducing signals.     

Estimating the selective advantage of mutant p53 tumour cells to repeated rounds of hypoxia

The tumour suppressor gene, p53, plays an important role in tumour development. Under low levels of oxygen (hypoxia), cells expressing wild-type p53 undergo programmed cell death (apoptosis), whereas cells expressing mutations in the p53 gene may survive and express angiogenic growth factors that stimulate tumour vascularization. Given that cells expressing mutations in the p53 gene have been observed in many forms of human tumour, it is important to understand how both wild-type and mutant cells react to hypoxic conditions. In this paper a mathematical model is presented to investigate the effects of alternating periods of hypoxia and normoxia (normal oxygen levels) on a population of wild-type and mutant p53 tumour cells. The model consists of three coupled ordinary differential equations that describe the densities of the two cell types and the oxygen concentration and, as such, may describe the growth of avascular tumours in vitro and/or in vivo. Numerical and analytical techniques are used to determine how changes in the system parameters influence the time at which mutant cells become dominant within the population. A feedback mechanism, which switches off the oxygen supply when the total cell density exceeds a threshold value, is introduced into the model to investigate the impact that vessel collapse (and the associated hypoxia) has on the time at which the mutant cells become dominant within vascular tumours growing in vivo. Using the model we can predict the time it takes for a subpopulation of mutant p53 tumour cells to become the dominant population within either an avascular tumour or a localized region of a vascular tumour. Based on independent experimental results, our model suggests that the mutant population becomes dominant more quickly in vivo than in vitro (12 days vs 17 days).     

p53 gene status and chemosensitivity in ovarian cancer.

Recent studies suggest that drug induced apoptosis relates to the sensitivity. p53 gene, which has a pivotal role in inducing apoptosis, frequently mutates in ovarian cancer. Therefore, p53 gene status and chemosensitivity in epithelial ovarian cancer is discussed. Nonresponders to chemotherapy had mutations of the p53 gene more frequently (83% for nonresponders vs. 16% for responders) in patients with epithelial ovarian cancer undergoing platinum-base chemotherapy. Apoptotic index was significantly greater in tumors with wild-type p53 gene than those without the gene. p53 gene transduction markedly enhanced the sensitivity to cisplatin (CDDP) and CDDP-induced apoptosis, but did not affect the sensitivity to paclitaxel (PTX) nor PTX-induced apoptosis in ovarian cancer cells without p53 gene. The combination treatment with a recombinant adenovirus carrying a wild-type p53 gene (AxCAp53) and CDDP significantly suppressed tumor growth of ovarian cancer cells with and without p53 gene, compared with a single treatment of either AxCAp53 or CDDP in ovarian cancer xenograft. Apoptotic index was significantly higher and proliferating cell nuclear antigen labeling index was relatively lower in an ovarian cancer xenograft without p53 gene receiving combination treatment, compared with a single treatment of either CDDP or AxCAp53, suggesting that the transduction of p53 gene induces apoptosis, but does not enhance the DNA repair system. A significant survival advantage was observed in the combination treatment compared with other treatments in carcinoma peritonitis models. In conclusion, p53 gene status contributes the sensitivity to CDDP in ovarian cancer. Additionally, combination treatment of p53 gene transduction and CDDP may be an effective therapeutic modality for ovarian cancer without wild-type p53 gene.     

Accumulation of wild-type p53 protein upon gamma-irradiation induces a G2 arrest-dependent immunoglobulin kappa light chain gene expression.

The exposure of cells to DNA-damaging agents leads to the accumulation of wild-type p53 protein. Furthermore, overexpression of the wild-type p53, mediated by transfection of p53-coding cDNA, induced cells to undergo apoptosis or cell differentiation. In this study we found that the gamma-irradiation that caused the accumulation of wild-type p53 in 70Z/3 pre-B cells induced, in addition to apoptosis, cell differentiation. This was manifested by the expression of the kappa light chain immunoglobulin gene that coincided with the accumulation of cells at the G2 phase. Overexpression of mutant p53 in 70Z/3 cells interferes with both differentiation and accumulation of cells at the G2 phase, as well as with apoptosis, which were induced by gamma-irradiation. Furthermore, the increment in the wild-type p53 protein level following gamma-irradiation was disrupted in the mutant p53 overproducer-derived cell lines. This suggests that mutant p53 may exert a dominant negative effect in all of these activities. Data presented here show that while p53-induced apoptosis is associated with the G1 checkpoint, p53-mediated differentiation, which may be an additional pathway to escape the fixation of genetic errors, may be associated with the G2 growth arrest phase.     

Abrogation of p53 by its antisense in MCF-7 breast carcinoma cells increases cyclin D1 via activation of Akt and promotion of cell proliferation

The p53 protein has been a subject of intense research interest since its discovery as about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancers suggesting a critical role of p53 in breast cancer development, growth and chemosensitivity. This report describes the derivation and characterization of MCF-7As53, an isogenic cell line derived from MCF-7 breast carcinoma cells in which p53 was abrogated by antisense p53 cDNA. Similar to MCF-7 and simultaneously selected hygromycin resistant MCF-7H cells, MCF-7As53 cells have consistent basal epithelial phenotype, morphology, and estrogen receptor expression levels at normal growth conditions. Present work documents investigation of molecular variations, growth kinetics, and cell cycle related studies in relation to absence of wild-type p53 protein and its transactivation potential as well. Even though wild-type tumor suppressor p53 is an activator of cell growth arrest and apoptosis-mediator genes such as p21, Bax, and GADD45 in MCF-7As53 cells, no alterations in expression levels of these genes were detected. The doubling time of these cells decreased due to depletion of G0/G1 cell phase because of constitutive activation of Akt and increase in cyclin D1 protein levels. This proliferative property was abrogated by wortmannin, an inhibitor of PI3-K/Akt signaling pathway. Therefore this p53 null cell line indicates that p53 is an indispensable component of cellular signaling system which is regulated by caveolin-1 expression, involving Akt activation and increase in cyclin D1, thereby promoting proliferation of breast cancer cells.     

The promise and obstacle of p53 as a cancer therapeutic agent

p53 is a tumor suppressor gene that is mutated in greater than 50% of human cancers. The action of p53 as a tumor suppressor involves inhibition of cell proliferation through cell cycle arrest and/or apoptosis. Loss of p53 function therefore allows the uncontrolled proliferation associated with cancerous cells. While design of most anti-cancer agents has focused on targeting and inactivating cancer promoting targets, such as oncogenes, recent attention has been given to restoring the lost activity of tumor suppressor genes. Because the loss of p53 function is so prevalent in human cancer, this protein is an ideal candidate for such therapy. Several gene therapeutic strategies have been employed in the attempt to restore p53 function to cancerous cells. These approaches include introduction of wild-type p53 into cells with mutant p53; the use of small molecules to stabilize mutant p53 in a wild-type, active conformation; and the introduction of agents to prevent degradation of p53 by proteins that normally target it. In addition, because mutant p53 has oncogenic gain of function activity, several approaches have been investigated to selectively target and kill cells harboring mutant p53. These include the introduction of mutant viruses that cause cell death only in cells with mutant p53 and the introduction of a gene that, in the absence of functional p53, produces a toxic product. Many obstacles remain to optimize these strategies for use in humans, but, despite these, restoration of p53 function is a promising anti-cancer therapeutic approach.