Person:

Little, John

Background: We have previously shown that in vitro radiosensitivity of human tumor cells segregate non-randomly into a limited number of groups. Each group associates with a specific genotype. However we have also shown that abrogation of a single gene (p21) in a human tumor cell unexpectedly sensitized xenograft tumors comprised of these cells to radiotherapy while not affecting in vitro cellular radiosensitivity. Therefore in vitro assays alone cannot predict tumor response to radiotherapy. In the current work, we measure in vitro radiosensitivity and in vivo response of their xenograft tumors in a series of human tumor lines that represent the range of radiosensitivity observed in human tumor cells. We also measure response of their xenograft tumors to different radiotherapy protocols. We reduce these data into a simple analytical structure that defines the relationship between tumor response and total dose based on two coefficients that are specific to tumor cell genotype, fraction size and total dose. Methods: We assayed in vitro survival patterns in eight tumor cell lines that vary in cellular radiosensitivity and genotype. We also measured response of their xenograft tumors to four radiotherapy protocols: 8 × 2 Gy; 2 × 5Gy, 1 × 7.5 Gy and 1 × 15 Gy. We analyze these data to derive coefficients that describe both in vitro and in vivo responses. Results: Response of xenografts comprised of human tumor cells to different radiotherapy protocols can be reduced to only two coefficients that represent 1) total cells killed as measured in vitro 2) additional response in vivo not predicted by cell killing. These coefficients segregate with specific genotypes including those most frequently observed in human tumors in the clinic. Coefficients that describe in vitro and in vivo mechanisms can predict tumor response to any radiation protocol based on tumor cell genotype, fraction-size and total dose. Conclusions: We establish an analytical structure that predicts tumor response to radiotherapy based on coefficients that represent in vitro and in vivo responses. Both coefficients are dependent on tumor cell genotype and fraction-size. We identify a novel previously unreported mechanism that sensitizes tumors in vivo; this sensitization varies with tumor cell genotype and fraction size.

There has been considerable recent interest in the mechanisms by which recessive mutations involving cancer genes may be expressed. We have developed an in vitro model to study this phenomenon in an endogenous autosomal gene in human cells. We have analyzed the molecular structural changes that lead to loss of heterozygosity at the thymidine kinase (tk) locus. The results indicate that expression of a recessive allele frequently occurs by loss of heterozygosity at that locus. Over 90% of spontaneous mutants at the tk locus arose by allele loss. The fraction of induced mutants that arose by this mechanism depended upon the inducing agent. Loss of the active tk allele was often accompanied by loss of linked genetic loci on the long arm of chromosome 17. These results suggest that large-scale chromosomal mutations resulting from events such as deletion or mitotic recombination may be an important mechanism for the expression of activated or mutated recessive genes in human cells. Such recessive mutations could involve oncogenes or other growth regulatory genes important in carcinogenesis.

We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

The importance of stress-induced p53 activation has been extensively investigated and well established. How the basal activity of p53 prevents carcinogenesis, however, remains incompletely understood. We report the identification of a novel p53 inhibitor, UXT, which binds to MDMX and suppresses the basal activity of p53. Interestingly, human TCGA database indicates that the UXT gene is frequently amplified in human sarcoma where p53 mutation is rare. We thus used sarcoma as a model to show that UXT acts as an oncogene promoting cell proliferation in vitro and tumor progression in vivo. A screening of 10 major cellular pathways uncovered that UXT-mediated p53 inhibition results in an activation of NF-κB, leading to induction of glycolysis. While elevated glycolytic metabolism provides growth advantage it also renders UXT expressing sarcoma cells heightened sensitivity to glycolysis inhibition. Altogether, our data demonstrate a crucial role for the basal activity of p53 in restriction of NF-κB. By impeding such an activity of p53, UXT unleashes the oncogenic activity of NF-κB resulting in induction of glycolysis fueling carcinogenesis.

Metabolic rewiring, specifically elevated glycolytic metabolism is a hallmark of cancer. Global chromatin structure regulates gene expression, DNA repair, and also affects cancer progression. But the interrelationship between tumor metabolism and chromatin architecture remain unclear. Here we show that increased glycolysis in cancer cells promotes an open chromatin configuration. Using complementary methods including Micrococcal nuclease (MNase) digestion assay, electron microscope and immunofluorescence staining, we demonstrate that glycolysis inhibition by pharmacological and genetic approaches was associated with induction of compacted chromatin structure. This condensed chromatin status appeared to result chiefly from histone hypoacetylation as restoration of histone acetylation with an HDAC inhibitor reversed the compacted chromatin state. Interestingly, glycolysis inhibition-induced chromatin condensation impeded DNA repair efficiency leading to increased sensitivity of cancer cells to DNA damage drugs, which may represent a novel molecular mechanism that can be exploited for cancer therapy.