Ferredoxin reductase affects p53-dependent, 5-fluorouracil-induced apoptosis in colorectal cancer cells.

Loss of p53 gene function, which occurs in most colon cancer cells, has been shown to abolish the apoptotic response to 5-fluorouracil (5-FU). To identify genes downstream of p53 that might mediate these effects, we assessed global patterns of gene expression following 5-FU treatment of isogenic cells differing only in their p53 status. The gene encoding mitochondrial ferredoxin reductase (protein, FR; gene, FDXR) was one of the few genes significantly induced by p53 after 5-FU treatment. The FR protein was localized to mitochondria and suppressed the growth of colon cancer cells when over-expressed. Targeted disruption of the FDXR gene in human colon cancer cells showed that it was essential for viability, and partial disruption of the gene resulted in decreased sensitivity to 5-FU-induced apoptosis. These data, coupled with the effects of pharmacologic inhibitors of reactive oxygen species, indicate that FR contributes to p53-mediated apoptosis through the generation of oxidative stress in mitochondria.

Requirement for p53 and p21 to sustain G2 arrest after DNA damage.

After DNA damage, many cells appear to enter a sustained arrest in the G2 phase of the cell cycle. It is shown here that this arrest could be sustained only when p53 was present in the cell and capable of transcriptionally activating the cyclin-dependent kinase inhibitor p21. After disruption of either the p53 or the p21 gene, gamma radiated cells progressed into mitosis and exhibited a G2 DNA content only because of a failure of cytokinesis. Thus, p53 and p21 appear to be essential for maintaining the G2 checkpoint in human cells.

Human cancer cells require ATR for cell cycle progression following exposure to ionizing radiation.

The vast majority of cancer cells have defective checkpoints that permit the cell cycle to progress in the presence of double-strand DNA breaks (DSBs) caused by ionizing radiation (IR) and radiomimetic drugs. ATR (ataxia telangiectasia-mutated and Rad3-related) has recently been shown to be activated by DSBs, although the consequences of this activity are largely unknown. In this report, we use advanced gene targeting methods to generate biallelic hypomorphic ATR mutations in human colorectal cancer cells and demonstrate that progression of the cancer cell cycle after IR treatment requires ATR. Cells with mutant ATR accumulated at a defined point at the beginning of the S phase after IR treatment and were unable to progress beyond that point, whereas cells at later stages of the S phase during the time of irradiation progressed and completed DNA replication. The prolonged arrest of ATR mutant cancer cells did not involve the ataxia telangiectasia mutated-dependent S-phase checkpoint, but rather closely resembled a previously characterized form of cell cycle arrest termed S-phase stasis. As ATR strongly contributed to clonogenic survival after IR treatment, these data suggest that blocking ATR activity might be a useful strategy for inducing S-phase stasis and promoting the radiosensitization of checkpoint-deficient cancer cells.

Disruption of p53 in human cancer cells alters the responses to therapeutic agents.

We have examined the effects of commonly used chemotherapeutic agents on human colon cancer cell lines in which the p53 pathway has been specifically disrupted by targeted homologous recombination. We found that p53 had profound effects on drug responses, and these effects varied dramatically depending on the drug. The p53-deficient cells were sensitized to the effects of DNA-damaging agents as a result of the failure to induce expression of the cyclin-dependent kinase inhibitor p21. In contrast, p53 disruption rendered cells strikingly resistant to the effects of the antimetabolite 5-fluorouracil (5-FU), the mainstay of adjuvant therapy for colorectal cancer. The effects on 5-FU sensitivity were observed both in vitro and in vivo, were independent of p21, and appeared to be the result of perturbations in RNA, rather than DNA, metabolism. These results have significant implications for future efforts to maximize therapeutic efficacy in patients with defined genetic alterations.

Cloning, expression, and chromosomal localization of the 140-kilodalton subunit of replication factor C from mice and humans.

We have isolated a full-length mouse cDNA encoding a lysine-rich protein of 1,131 amino acids with a calculated molecular mass of 126 kDa. The protein binds in a sequence-unspecific manner to DNA, is localized exclusively in the nucleus, and contains a putative ATP binding site and a stretch of 80 amino acids with homology to the carboxy terminus of prokaryotic DNA ligases. On the basis of the following facts, we conclude that the isolated cDNA encodes the 140-kDa subunit of mouse replication factor C (mRFC140). (i) The sequence around the ATP binding site shows significant homology to three small subunits of human replication factor C. (ii) Polyclonal antibodies raised against the protein encoded by this cDNA cross-react with the 140-kDa subunit of purified human replication factor C (hRFC140) and recognize in mouse cell extracts an authentic protein with an apparent molecular mass of 130 kDa. (iii) Sequence comparison with a human cDNA isolated by using tryptic peptide sequence information from purified hRFC140 revealed 83% identity of the encoded proteins. The mRFC140 gene is ubiquitously expressed, and two mRNAs approximately 5.0 and 4.5 kb long have been detected. The gene was mapped by in situ hybridization to mouse chromosome 5, and its human homolog was mapped to chromosome 4 (p13-p14).

Cell death and cancer therapy.

Successful cancer therapy requires the selective killing of cancer cells. Many molecular components of the pathways that lead to cell death have recently been identified and a number of these, including p53, Apaf-1, and members of the inhibitor of apoptosis protein and Bcl-2 gene families, have been found to be altered or disregulated in many cancers. These recent advances and the ongoing elucidation of how these pathways work is providing clues as to how therapeutically resistant cancers might be attacked.

ATR mediates cisplatin resistance in a p53 genotype-specific manner.

The protein kinase encoded by the ataxia telangiectasia and Rad3-related (ATR) gene is activated by DNA-damaging agents that are frequently used as anticancer therapeutics. Inhibition of ATR expression in cultured cancer cells has been demonstrated to increase sensitivity to chemotherapeutic drugs, including the DNA-crosslinking agent cisplatin. Cisplatin is a widely used and effective drug, but its use is associated with significant toxicity. Here, we demonstrate that genetic inhibition of ATR expression selectively enhanced cisplatin sensitivity in human colorectal cancer cells with inactivated p53. A knock-in strategy was used to restore wild-type p53 in cells harboring wild-type or mutant ATR alleles. Knock-in of functional p53 in ATR-deficient cells restored checkpoint function, suppressed apoptotic pathways and markedly increased clonogenic survival after cisplatin treatment. These results suggest that a strategy that combines specific inhibitors of ATR and conventional therapies might promote synthetic lethality in p53-deficient tumors, and thus minimize toxicity to normal tissues.

cDNAs encoding the large subunit of human replication factor C.

Replication factor C (RFC) is a multisubunit, DNA polymerase accessory protein required for the coordinated synthesis of both DNA strands during simian virus 40 DNA replication in vitro. Previous studies have shown that RFC is a DNA-dependent ATPase that binds in a structure-specific manner to the 3' end of a primer hybridized to a template DNA, an activity thought intrinsic to the 140-kDa component of this multisubunit complex. Here, the isolation and analysis of cDNAs encoding this subunit is described. Analysis of the full-length coding sequence revealed an open reading frame of 3.4 kb, encoding an 1148-amino acid protein with a predicted molecular mass of 130 kDa. A putative ATP-binding motif was observed that is similar to a motif in several of the smaller subunits of RFC and in functionally homologous replication factors of bacterial and viral origin. A "DEAD" box is also conserved among these proteins. The predicted protein shows significant identity with a DNA-binding protein of murine origin (B. Luckow, P. Lichter, and G. Schütz, personal communication). Regions of similarity were also seen between the amino acid sequences of the 140-kDa subunit of RFC, poly(ADP-ribose) polymerase, and bacterial DNA ligases--possibly representing a conserved structural feature of these proteins that bind similar DNA substrates.

Securin is required for chromosomal stability in human cells.

Abnormalities of chromosome number are the most common genetic aberrations in cancer. The mechanisms regulating the fidelity of mitotic chromosome transmission in mammalian cells are therefore of great interest. Here we show that human cells without an hSecurin gene lose chromosomes at a high frequency. This loss was linked to abnormal anaphases during which cells underwent repetitive unsuccessful attempts to segregate their chromosomes. The abnormal mitoses were associated with biochemical defects in the activation of separin, the sister-separating protease, rendering it unable to cleave the cohesin subunit Scc1 efficiently. These results illuminate the function of mammalian securin and show that it is essential for the maintenance of euploidy.

Targeted deletion of Smad4 shows it is required for transforming growth factor beta and activin signaling in colorectal cancer cells.

Smad4 (DPC4) is a candidate tumor suppressor gene that has been hypothesized to be critical for transmitting signals from transforming growth factor (TGF) beta and related ligands. To directly test this hypothesis, the Smad4 gene was deleted through homologous recombination in human colorectal cancer cells. This deletion abrogated signaling from TGF-beta, as well as from the TGF-beta family member activin. These results provide unequivocal evidence that mutational inactivation of Smad4 causes TGF-beta unresponsiveness and provide a basis for understanding the physiologic role of this gene in tumorigenesis.