Complementation of two mutant p53: Implications for loss of heterozygosity in cancer

AbstractRemarkably, a cancer cell rarely possesses two mutant p53 proteins. Instead, mutation of one allele is usually associated with loss of the second p53 allele. Why do not two mutant p53 co-exist? We hypothesize that two different p53 may complement each other, when expressed at equal levels. By titrating trans-deficient and DNA-binding-deficient p53 in cells with mutant p53 and by co-transfecting distinct mutant p53 in p53-null cells, we demonstrated activation of p53-dependent transcription. We suggest that, due to complementation of two mutant p53, cancer cells need to delete the second p53 allele rather than mutate it

The Role of Wild-Type p53 in Cisplatin-Induced Chk2 Phosphorylation and the Inhibition of Platinum Resistance with a Chk2 Inhibitor

The major obstacle in platinum chemotherapy is the repair of platinum-damaged DNA that results in increased resistance, reduced apoptosis, and finally treatment failure. Our research goal is to determine and block the mechanisms of platinum resistance. Our recent studies demonstrate that several kinases in the DNA-repair pathway are activated after cells are exposed to cisplatin. These include ATM, p53, and Chk2. The increased Chk2 phosphorylation is modulated by p53 in a wild-type p53 model. Overexpression of p53 by cDNA transfection in wt-p53 (but not p53 deficient) cells doubled the amount of Chk2 phosphorylation 48 hours after cisplatin treatment. p53 knockdown by specific siRNA greatly reduced Chk2 phosphorylation. We conclude that wild-type p53, in response to cisplatin stimulation, plays a role in the upstream regulation of Chk2 phosphorylation at Thr-68. Cells without normal p53 function survive via an alternative pathway in response to the exogenous influence of cisplatin. We strongly suggest that it is very important to include the p53 mutational status in any p53 involved studies due to the functional differentiation of wt p53 and p53 mutant. Inhibition of Chk2 pathway with a Chk2 inhibitor (C3742) increased cisplatin efficacy, especially those with defective p53. Our findings suggest that inhibition of platinum resistance can be achieved with a small-molecule inhibitor of Chk2, thus improving the therapeutic indices for platinum chemotherapy

Neutral evolution of drug resistant colorectal cancer cell populations is independent of their KRAS status

Emergence of tumor resistance to an anti-cancer therapy directed against a putative target raises several questions including: (1) do mutations in the target/pathway confer resistance? (2) Are these mutations pre-existing? (3) What is the relative fitness of cells with/without the mutation? We addressed these questions in patients with metastatic colorectal cancer (mCRC). We conducted an exhaustive review of published data to establish a median doubling time for CRCs and stained a cohort of CRCs to document mitotic indices. We analyzed published data and our own data to calculate rates of growth (g) and regression (d, decay) of tumors in patients with CRC correlating these results with the detection of circulating MT-KRAS DNA. Additionally we estimated mathematically the caloric burden of such tumors using data on mitotic and apoptotic indices. We conclude outgrowth of cells harboring intrinsic or acquired MT-KRAS cannot explain resistance to anti-EGFR (epidermal growth factor receptor) antibodies. Rates of tumor growth with panitumumab are unaffected by presence/absence of MT-KRAS. While MT-KRAS cells may be resistant to anti-EGFR antibodies, WT-KRAS cells also rapidly bypass this blockade suggesting inherent resistance mechanisms are responsible and a neutral evolution model is most appropriate. Using the above clinical data on tumor doubling times and mitotic and apoptotic indices we estimated the caloric intake required to support tumor growth and suggest it may explain in part cancer-associated cachexia

Acetylation Of Pronase Guanidine Stable Chymoelastase

Acetylation of Pronase Guanidine Stable Chymoelastase in 50% glycerol results in modification of both the (alpha)-amino group of isoleucine 1 and the (epsilon)-amino group of the single lysine residue with retention of activity. The pH-rate profile of inhibition with chloromethylketones is also unchanged.The group in chymotrypsin that governs the activity with specific substrates at alkaline pH is believed to be the amino group of the amino-terminal isoleucine. In order to re-examine the role of the amino group in chymotrypsin activity we first attempted chemical modification with acetic anhydride, a technique that has been used extensively in elucidating the role of various groups in proteins, including chymotrypsin. We have found that glycerol prevents the loss of activity upon acetylation by protecting the alpha amino group. Modification of the amino group with methylacetimidate has been shown to abolish the activity of delta chymotrypsin. Guanidination of lysine amino groups prior to amidination protects against this loss.The role of glycerol or guanidination in stabilizing chymotrypsin has been shown in studies with proflavin to be exerted through a shift in the equilibrium between the active and inactive forms of the enzyme so that at any pH the fraction of enzyme in the active conformation is increased.The pKa of the alpha amino group depends on the extent to which it is buried in a salt bridge, thus glycerol or guanidination provide a way of modifying the alpha amino group. Their effect on the titration behavior of this group has been studied. These techniques can be used to shift the apparent pKa of the alpha amino group, allowing one to study the importance of this group in the activity of chymotrypsin at alkaline pH. Our data indicate that the alpha amino group may not be completely responsible for the loss of activity of chymotrypsin at alkaline pH