Cancer research has previously focused on the identification of specific genes and pathways responsible for cancer initiation and progression based on the prevailing viewpoint that cancer is caused by a stepwise accumulation of genetic aberrations. This viewpoint, however, is not consistent with the clinical finding that tumors display high levels of genetic heterogeneity and distinctive karyotypes. We show that chromosomal instability primarily generates stochastic karyotypic changes leading to the random progression of cancer. This was accomplished by tracing karyotypic patterns of individual cells that contained either defective genes responsible for genome integrity or were challenged by onco-proteins or carcinogens that destabilized the genome. Analysis included the tracing of patterns of karyotypic evolution during different stages of cellular immortalization. This study revealed that non-clonal chromosomal aberrations (NCCAs) (both aneuploidy and structural aberrations) and not recurrent clonal chromosomal aberrations (CCAs) are directly linked to genomic instability and karyotypic evolution. Discovery of "transitional CCAs" during in vitro immortalization clearly demonstrates that karyotypic evolution in solid tumors is not a continuous process. NCCAs and their dynamic interplay with CCAs create infinite genomic combinations leading to clonal diversity necessary for cancer cell evolution. The karyotypic chaos observed within the cell crisis stage prior to establishment of the immortalization further supports the ultimate importance of genetic aberrations at the karyotypic or genome level. Therefore, genomic instability generated NCCAs are a key driving force in cancer progression. The dynamic relationship between NCCAs and CCAs provides a mechanism underlying chromosomal based cancer evolution and could have broad clinical applications.

ABBREVIATIONS DNMTsDNA Methyltransferases GST glutathione-S-transferase ACKNOWLEDGEMENTSWe thank Drs.Tom Shenk for GST-p53 fusion constructs, Chih-Lin Hsieh for Dnmt3a and the Dnmt3am expression constructs, and Bert Vogelstein for the WWP-luciferase construct. This research was partly supported by Grants from Department of Defense (DAMD17-02-1-0619) and the NIH (P30 ES06639 and RO1 CA89526). Research PaperDNA Methyltransferase-3a Interacts with p53 and Represses p53-Mediated Gene Expression ABSTRACTGenome stability maintenance is regulated by both genetic and epigenetic mechanisms. DNA methylation is the predominant epigenetic mechanism in regulation of gene expression and in suppression of mobile DNA elements from random integration in the genome. The importance of DNA methylation in tumorigenesis has been demonstrated in cancer cells, which harbor global genomic DNA hypomethylation and regional hypermethylation at CpG islands of tumor suppressor genes. DNA methylation is mediated by a class of DNA methyltransferases (Dnmts) involved in de novo methylation of genomic DNA and in the maintenance of DNA methylation patterns during replication. Global genomic DNA demethylation induced by 5-Aza-deoxycytidine activates the p53 signaling pathway and induces apoptosis, suggesting that DNA methylation mediated by Dnmts is associated with p53 signaling in maintaining genome stability. In this report, we show that Dnmt3a interacts with p53 directly and represses p53-mediated transactivation of the p21 gene. It was found that trans-repression by Dnmt3a does not require the methyltransferase activity implying that transcriptional repression does not involve promoter silencing through DNA methylation by Dnmt3a. Finally, the activity of Dnmt3a in vivo was demonstrated when this enzyme was overexpressed in a breast cell line in which Dnmt3a repressed p21 upregulation following DNA damage. The results presented in this study provide new understanding of tumor promotion as mediated by Dnmt3a through its interaction with p53, and suppression of the p53-mediated transcription of tumor suppressor genes. Given that the expression of Dnmts is increased in certain cancers, it is likely that increased Dnmts could block the transactivation function of p53 following its induction by chemotherapeutic drugs resulting in chemoresistance. The use of a DNA methyltransferase inhibitor would therefore restore the p53 tumor suppression function and the utilization of such an inhibitor in combination with DNA damage agents might be an effective therapy for certain cancers.

DNA damage can lead to either DNA repair with cell survival or to apoptotic cell death. Although the biochemical processes underlying DNA repair and apoptosis have been extensively studied, the mechanisms by which cells determine whether the damage will be repaired or the apoptotic pathway will be activated is largely unknown. We have studied the role of nucleotide excision repair (NER) in cisplatin DNA damage-induced apoptotic cell death using both normal human fibroblasts and NERdefective xeroderma pigmentosum (XP) XPA and XPG cells. It also showed that a functional XPC protein was required for the association of the ATM protein to genomic DNA. These results suggest that the NER process may prevent the cisplatin treatment-induced apoptosis by activating the ATM protein, and that the presence of the XPC protein is essential for recruiting the ATM protein to the DNA template.Many anticancer drugs are targeted to the genomic DNA of cancer cells to generate DNA damage and block DNA replication and/or gene transcription, resulting in cell cycle arrest and apoptotic cell death (apoptosis). However, cancer cells can avoid this DNA damage-induced cell death through several mechanisms. DNA repair is one of the most important mechanisms that prevent DNA damage-induced cell death (1). Although many studies have been done regarding DNA repair and apoptosis (2-7), the molecular mechanism that determines whether the damage will be repaired or that the damaged cells will undergo apoptosis is largely unknown. The lack of such knowledge has significantly limited our understandings of cancer cell drug resistance and hindered our abilities in the design and development of new drugs for effective cancer treatment.Nucleotide excision repair (NER) 2 is the major DNA repair pathway utilized in the repair of bulky DNA damage generated by most environmental insults and therapeutic drugs (1,8,9). The NER process is initiated by DNA damage recognition and the binding of the XPC-HR23B complex to damaged DNA (10 -14), which further recruits other NER components including XPA, TFIIH, XPG, and XPF/ERCC1 to the damaged site (12,13,15,16). The XPG protein makes a 3Ј incision, which is followed by a 5Ј incision made by the XPF/ERCC1, resulting in a singlestranded gap of 27-32 nucleotides (17). The DNA polymerases (pol ⑀ or pol ␦) fill the gap and the DNA ligase seals the gap to complete the DNA repair process. Interestingly, defects in most of the NER proteins, including XPA, XPB, XPD, XPF and XPG, lead to elevated sensitivities of the cells to many DNA damaging reagents. However, defects of XPC and XPE proteins do not cause increased sensitivity of the cells to DNA damaging treatment (18). Therefore, studying the DNA damage-mediated signaling process in these NER-defective cells will provide important insights into the mechanism of DNA repair in preventing DNA damage-induced apoptosis.DNA damage also promotes cell cycle checkpoint regulation. Both ATM and ATR proteins play important roles in DNA damage-induced cell cycle checkpoint regulation...