Tapas Mukhopadhyay

Retrovirus-mediated wild-type p53 gene transfer to tumors of patients with lung cancer

A retroviral vector containing the wild–type p53 gene under control of a β–actin promoter was produced to mediate transfer of wild–type p53 into human non–small cell lung cancers by direct injection. Nine patients whose conventional treatments failed were entered into the study. No clinically significant vector–related toxic effects were noted up to five months after treatment. In situ hybridization and DNA polymerase chain reaction showed vector–p53 sequences in posttreatment biopsies. Apoptosis (programmed cell death) was more frequent in posttreatment biopsies than in pretreatment biopsies. Tumor regression was noted in three patients, and tumor growth stabilized in three other patients.

Induction of apoptosis in human lung cancer cells after wild-type p53 activation by methoxyestradiol

2-methoxyestradiol (2-MeOE2) treatment caused significant growth inhibition of H460 and A549 human lung cancer cell lines which contain wild-type p53. However, 2-MeOE2 had a little effect on the p53 negative H358 and p53 mutated H322 cell lines. Western blot analysis indicated that 2-MeOE2 treatment resulted in an eightfold increase in the endogenous wild-type p53 protein, while the level of the mutant p53 protein remained unchanged. TdT staining indicated that following 2-MeOE2-mediated increases in wildtype p53 protein, cells bypass the G1-S checkpoint of the cell cycle with 30 to 40% undergoing apoptosis. Introduction of anti-sense wt-p53 into wt-p53 cells abrogated the 2-MeOE2 effect. A significant portion of lung cancer retains the wild-type p53 gene therefore, 2-MeOE2 may have therapeutic application.

Superinduction of wild-type p53 protein after 2-methoxyestradiol treatment of Ad5p53-transduced cells induces tumor cell apoptosis

Because 2-methoxyestradiol (2-MeOE2) induces and stabilizes wild-type p53 protein (wt p53) in human lung cancer cell lines posttranscriptionally, we sought to study its effects on Ad5p53-transduced lung cancer cell lines at a low multiplicity of infection (1 MOI). Treating these cells with 2-MeOE2 resulted in superinduction of wt p53 protein expression followed by apoptosis, as shown by terminal deoxynucleotidyl transferase (TdT) staining, and upregulation of wt p53 expression, as shown by Western blot analysis. When transduced with Ad5p53 alone at 1 MOI, the cell lines grew rapidly. Moreover, adenoviral-vector-mediated p53 gene transfer followed by 2-MeOE2 treatment caused 80% growth inhibition in the cell lines regardless of their p53 status. Thus, p53 superinduction and apoptosis after 2-MeOE2 treatment in Ad5p53-transduced cells appears to be a unique strategy with significant implications for cancer gene therapy.

Cellular and humoral immune responses to adenovirus and p53 protein antigens in patients following intratumoral injection of an adenovirus vector expressing wild-type p53 (Ad-p53)

The immune responses of 10 patients with advanced non-small cell lung cancer receiving monthly intratumoral injections of a recombinant adenovirus containing human wild-type p53 (Ad-p53) to adenovirus and transgene antigens were studied. The predominate cellular and humoral immune responses as measured by lymphocyte proliferation and neutralizing antibody (Ab) formation were to adenovirus serotype 5 vector antigens, with increased responses in posttreatment samples. Consistent alterations in posttreatment cellular and humoral immune responses to p53 epitopes were not observed, and cytotoxic Abs to human lung cancer cells were not generated. Patients in this study had evidence of an antitumoral effect of this treatment with prolonged tumor stability or regression; however, neither Abs to p53 protein nor increased lymphocyte proliferative responses to wild-type or mutant p53 peptides have been consistently detected.

5-aza-2′-deoxycytidine upregulates caspase-9 expression cooperating with p53-induced apoptosis in human lung cancer cells

Treating lung cancer cell lines using low-dose 5-aza-2′-deoxycytidine (DAC) caused an accumulation of procaspase-9 through mRNA upregulation, but the cells did not undergo apoptosis. However, when cells were treated with DAC and infected with a low dose of a recombinant wild-type p53 adenovirus vector (Ad-p53), a synergistic growth inhibitory effect was observed. Combination treatment induced Apaf-1 and procaspase-9 expression in which cytochrome c releases by Ad-p53 triggered the mitochondrial pathway of apoptosis. Selective blockage of caspase-9 activities by Z-LEHD-FMK completely attenuated DAC-induced enhancement of apoptosis mediated by Ad-p53 infection, and ectopic overexpression of procaspase-9 sensitized cells to Ad-p53-induced apoptosis in p53-null cells. In addition, DAC sensitized lung cancer cells to cisplatin and paclitaxel. Induction of the mitochondrial pathway of apoptosis using a slightly toxic dose of DAC may therefore be a strategy for treating lung cancer, and DAC treatment may have clinical implications when combined with chemotherapy or apoptosis-inducing gene therapy.

ORAL ADMINISTRATION OF AN ESTROGEN METABOLITE-INDUCED POTENTIATION OF RADIATION ANTITUMOR EFFECTS IN PRESENCE OF WILD-TYPE p53 IN NON-SMALL-CELL LUNG CANCER

PURPOSE The purpose of this study was to investigate the efficacy of 2-methoxyestradiol as an antitumor and radiosensitizing agent for the treatment of human malignancy. METHODS AND MATERIALS Two cancer cell lines with wild-type p53 status were exposed first to irradiation and then to an oral formulation of the nontoxic metabolite 2-methoxyestradiol (2ME) to stabilize p53 levels. RESULTS Cell growth was inhibited via G1 growth and apoptosis. Subsequent in vitro growth and Tunel assays indicated that this combination was superior to radiation alone at inducing p53 protein accumulation, stabilizing p53 protein levels, and substantially reducing long-term tumor cell growth (approximately 80%) and colony formation (approximately 95%) in vitro, and inducing apoptosis. However, harboring mutated p53, H322 cell line, was relatively insensitive to such a treatment regimen. Western blot analysis revealed that growth inhibition was associated with increased levels of p53 and p21 protein accumulation. Experiments with subcutaneous tumor in a nu/nu mouse showed the combination treatment to be superior to radiation alone at reducing tumor growth ( approximately 50% reduction as compared to radiation alone) in vivo. CONCLUSION Thus, our studies confirmed a unique strategy whereby oral administration of a nontoxic estrogen metabolite, 2ME, significantly enhanced the radiation effect on a subcutaneous tumor without any toxicity and suggesting that this strategy may be clinically useful as an adjuvant therapy.

p53 Suppressor Gene

1. The Role of p53 in Cancer.- 2. Gene Structure.- 3. Wild-Type versus Mutant p53.- 4. Biophysical and Biochemical Properties of the p53 Protein.- 5. Regulation and Modulation of the Function of p53.- 6. Potential Clinical Significance of the p53 Tumor Suppressor Gene in Cancer Patients.

Reduced telomeric signals and increased telomeric associations in human lung cancer cell lines undergoing p53-mediated apoptosis

Transduction of a p53-negative H1299 human non-small cell lung cancer cell line with an adenoviral vector containing wild-type p53 (Ad5p53) induced apoptosis. Analysis of the Ad5p53-infected H1299 cells showed high levels of telomeric association prior to apoptotic nuclear fragmentation. Similar telomeric association was observed in stably transfected clones of the wtH226b cell line, which expressed exogenous wild-type p53 protein and also showed complex chromosomal abnormalities including dicentrics, rings and fragments. Fluorescence in situ hybridization (FISH) analysis using a human telomeric DNA probe indicated reductions in telomere signals in both Ad5p53-infected H1299 cells and wtH226b-S cells. In contrast, stably transfected wtH226b-AS clones expressing antisense p53 cDNA showed no telomeric association and had high levels of telomeric signals associated with a faster growing phenotype. These results suggest that wild-type p53 is involved in shortening telomeres, a possibly early event in the p53-mediated apoptotic process and in the subsequent telomeric association that predisposes a cell to genetic instability and DNA fragmentation resulting in apoptotic cell death. Moreover, loss of telomeric signals may indicate a cells decision to undergo programmed cell death and, if so, could, serve as a sensitive marker of p53-mediated apoptosis.

C-terminus of p53 is required for G2 arrest

Mutation of four lysine residues in the p53 C-terminal domain inhibits MDM2-dependent ubiquitination of p53 and alters its subcellular distribution. This implies that modification (such as acetylation and phosphorylation) of amino acid residues in p53 C-terminal domain, regulate the biological functions of p53. In this study, we demonstrated that p53 with lysine residues 372, 373, 381, and 382 mutated to alanine (the A4 mutant) retained the transactivation activity of wild-type p53, although the transactivation activity of p21 promoter by the A4 mutant was slightly reduced. The inducible expression of wild-type p53 and the A4 mutant in H1299 cells caused growth inhibition due to cell-cycle arrest. Consistent with previous studies, the expression of wild-type p53 elicited G1 and G2 arrests. However, the cells expressing the A4 mutant underwent G1 arrest but not G2 arrest. Cyclin B1-associated kinase activity was reduced in cells expressing wild-type p53 but not A4, when the cells underwent G2 arrest. This suggests that modification of the p53 C-terminal domain might inhibit p53-mediated G2 arrest. In other words, p53 requires an intact C-terminus to induce G2 arrest.

Multiple lysine mutations in the C-terminus of p53 make it resistant to degradation mediated by MDM2 but not by human papillomavirus E6 and induce growth inhibition in MDM2-overexpressing cells

We have recently shown that lysine mutations in p53s putative C-terminal acetylation sites result in increased stability and cytoplasmic distribution of the p53 protein in a human lung cancer cell line. In the present study, we showed that when lysine residues 372, 373, 381, and 382 of p53 were substituted with alanine, the resulting A4 protein was resistant to MDM2-mediated proteosomal degradation but was highly sensitive to human papillomavirus E6-mediated proteolysis. When A4 and wild-type p53 were transfected into MDM2-overexpressing MCF-7 cells, A4 significantly reduced colony formation in vitro, when compared with wild-type p53. Our results suggest that A4 exerts a growth-inhibitory effect more efficiently than wild-type p53 does in cell lines that overexpress MDM2 and may therefore be a better therapeutic tool than wild-type p53 for certain cancers in which MDM2 is amplified or overexpressed.