The administration schedule of cyclin-dependent kinase inhibitor gene therapy and etoposide chemotherapy is a major determinant of cytotoxicity.

Cyclin-dependent kinase inhibitors are potent suppressors of cell growth and have been proposed as targets for gene replacement therapy in cancer. Expression of either p16INK4a or p21WAF1 protected cells from the cytotoxic effects of the topoisomerase II inhibitor, etoposide. A lower level of p53 was induced in CDK inhibitor-expressing etoposide-exposed cells suggesting that protection may be due to lower levels of DNA damage in the growth arrested cells. Exposure of human osteosarcoma cells to either p16INK4a or p21WAF1 prior to and during etoposide therapy protected cells against etoposide-induced cell death. Infection of the cells by Ad-p16INK4a or Ad-p21WAF1 following exposure to etoposide resulted in loss of the protective effect with evidence of enhanced growth inhibition. The results suggest that the schedule of administration of DNA damaging etoposide chemotherapy and cell cycle inhibitory therapy is a major determinant of the resulting cytotoxicity.

p73beta, unlike p53, suppresses growth and induces apoptosis of human papillomavirus E6-expressing cancer cells.

Human papillomavirus (HPV) is the major cause of cervical cancer worldwide. HPV-E6 protein targets the p53 tumor suppressor protein for degradation by ubiquitin-mediated proteolysis making such cancers resistant to p53-gene therapy. Here we show that infection of human cancer cells by E6-expressing adenovirus (Ad-E6) leads to degradation of both wild-type or mutant p53 protein. Interestingly, the p53-homologue candidate tumor suppressor p73 is not degraded in Ad-E6 infected cancer cells. Wild-type p73beta and not wild-type p53 or mutant p73 is a potent inhibitor of cancer colony growth and inducer of apoptosis, despite HPV-E6 overexpression. The results suggest a novel strategy using p73beta in gene therapy of HPV-E6 expressing cancers.

Bypass of abnormal MDM2 inhibition of p53-dependent growth suppression.

Oncoprotein MDM2 inhibits p53-dependent cell cycle arrest and apoptosis. MDM2-overexpressing human cancer cell lines (n = 3) were found to be resistant to growth inhibition after infection by p53-expressing adenovirus (Ad-p53), as compared to low MDM2-expressing tumors (n = 3), in vitro. The growth of MDM2-overexpressing tumors, however, was inhibited by p21-expressing adenovirus (Ad-p21) infection, and the cyclin-dependent kinase-inhibitory region of p21 was sufficient to bypass the MDM2-p53 feedback loop. The phosphorylation state of Rb correlated with the response to either p53 or p21 gene therapy. MDM2-overexpressing cancer cells infected by Ad-p21 also developed a quiescent large-cell morphology. The results suggest that MDM2-mediated resistance to p53 may be bypassed by p21 and that the Rb phosphorylation state may predict the effects on growth after Ad-p53 or Ad-p21 infection.

Defects in p21WAF1/CIP1, Rb, and c-myc signaling in phorbol ester-resistant cancer cells.

Growth arrest and differentiation of leukemic cells by phorbol 12-myristate 13-acetate (PMA) is accompanied by p53-independent activation of p21WAF1/CIP1 and c-myc down-regulation. We show that despite p21 induction in 7 of 12 human cancer cell lines treated with PMA, growth inhibition was observed only in two cell lines (SKBr3 breast and LNCaP prostate cancer cells). Treatment of SKBr3 and LNCaP cells with PMA was followed by Raf-1 hyperphosphorylation, p21 induction, Rb hypophosphorylation, c-myc down-regulation and growth inhibition. The 10 remaining PMA-resistant cell lines were comprised of 5 that failed to induce p21 and 5 that induced p21 but had defects in steps putatively downstream of this (Rb hypophosphorylation and c-myc down-regulation). Exogenous expression and subsequent failure to down-regulate c-myc protein expression in SKBr3 and LNCaP cells was correlated with acquisition of resistance to the growth inhibitory effect of PMA. Exogenous p21 expression down-regulated c-myc protein in PMA-sensitive cancer cells. Our findings suggest that induction of p21 and down-regulation of c-myc may be necessary steps in a PMA-induced growth-inhibitory pathway in cancer cells.

Single substitutions of phosphorothioates in the HDV ribozyme G73 define regions necessary for optimal self-cleaving activity.

Phosphorothioate (NTPalphaS) analogues were incorporated into the HDV genomic ribozyme by transcription with T7 polymerase. The introduction of a sulfur in place of the pro-Rp oxygen at the phosphate 5'to positions A64, A63, A43, U27, G62, C61, C44, C41, C22and C21appeared to inhibit self-cleavage activity of the G73 genomic ribozyme. Except for position C22, elevated levels of Mg2+rescued the reaction to various extents. When the sites were identified in the RNA sequence, they were clustered in three distinct regions that, in the secondary structure models, are predicted to be primarily single-stranded. Two of these regions have been proposed to form extensive interactions that are thought to involve a homopurine base pair. The third region is thought to be directly associated with assembly of the cleavage site.

Suppression of cancer cell growth by adenovirus expressing p21(WAF1/CIP1) deficient in PCNA interaction.

p53 tumor suppression is deficient in the majority of human cancers. Efforts to understand this pathway have identified cyclin-dependent kinase (CDK) inhibitors and suggested a potential for their replacement in human cancer. In the present studies, expression of a C-terminal deletion mutant of the human p21(WAF1/CIP1) CDK inhibitor completely suppressed the growth of colon cancer cells, whereas full-length p21 only partially suppressed growth. We prepared a replication-deficient adenoviral recombinant which expresses the p21 C-terminal mutant (Ad-WAF1-341) and compared its tumor suppressive abilities with Ad-p53 and Ad-LacZ. Ad-WAF1-341- and Ad-p53-infected cancer cells, but not Ad-LacZ-infected cancer cells, expressed a nuclear protein recognized by anti-p21 antibody and were deficient in cell cycle progression. The exogenous p21 mutant interacted with CDK2 but not proliferating cell nuclear antigen following infection of p21-/- cancer cells. Ad-WAF1-341 was more potent than Ad-p53 in inhibiting DNA synthesis in human papillomavirus 16 E6-expressing cancer cells. Most importantly, the Ad-WAF1-341-infected E6-expressing cells died, whereas most of the Ad-p53-infected cells continued to proliferate. Endonucleolytic cleavage of DNA was observed in Ad-WAF1-341-infected cancer cells. These observations suggest that Ad-WAF1-341 should be evaluated in the treatment of human papillomavirus-associated neoplasia and other neoplasias resistant to p53.