Most mutations after DNA damage in yeast Saccharomyces cerevisiae are induced by error-prone translesion DNA synthesis employing scRev1 and DNA polymerase that consists of scRev3 and scRev7 proteins. Recently, the human REV1 (hREV1) and REV3 (hREV3) genes were identified, and their products were revealed to be involved in UV-induced mutagenesis, as observed for their yeast counterparts. Human REV7 (hREV7) was also cloned, and its product was found to interact with hREV3, but the biological function of hREV7 remained unknown. We report here the analyses of precise interactions in the human REV proteins. The interaction between hREV1 and hREV7 was identified by the yeast two-hybrid library screening using a bait of hREV7, which was confirmed by in vitro and in vivo binding assays. The homodimerization of hREV7 was also detected in the two-hybrid analysis. In addition, the precise domains for interaction between hREV7 and hREV1 or hREV3 and for hREV7 homodimerization were determined. Although hREV7 interacts with both hREV1 and hREV3, a stable complex formation of the three proteins was undetectable in vitro. These findings suggest the possibility that hREV7 might play an important role in regulating the enzymatic activities of hREV1 and hREV3 for mutagenesis in response to DNA damage.An error-free DNA replication system is required to pass accurate genetic information on to the next generation. However, various kinds of DNA damage induced by endogenous and exogenous factors impair this replication ability and cause genetic alterations, resulting in cancer predisposition (1). Cells have excellent systems for avoiding these genetic alterations by removing and repairing the damaged lesions before DNA replication for maintaining the genetic stability of the organism; these systems include base excision repair, nucleotide excision repair, mismatch repair, and recombination repair (2, 3). If a lesion on template DNA escapes these repair systems, a polymerase may stall at this point and start synthesis again downstream, resulting in a single strand gap in the DNA, which can be repaired by postreplication repair. Usually, recombination repair in postreplication repair can fix this gap without base substitution, but when this repair does not happen, DNA synthesis by a bypass formation across the lesion called translesion synthesis (TLS) 1 may take place to fill the gap. This TLS may be held in the last resort for DNA repair because mutations can be induced during this step (for reviews, see Refs. 4 -6).In budding yeast Saccharomyces cerevisiae, the scRAD30 gene, the product of which is DNA polymerase , is involved in the error-free TLS that can replicate DNA through cis-syn thymine-thymine (T-T) dimer in an error-free manner (7-10), whereas the scREV1, scREV3, and scREV7 genes are involved in the error-prone TLS that frequently induces mutations at the damaged lesions (for reviews, see . It is known that most mutations induced after UV irradiation are caused by the products of these three REV genes. scRev1 protein is a ter...

To examine whether globotriaosylceramide (Gb3/CD77) is a receptor for verotoxins (VTs) in vivo, sensitivity of Gb3/CD77 synthase null mutant mice to VT-2 and VT-1 was analyzed. Although wild-type mice died after administration of 0.02 g of VT-2 or 1.0 g of VT-1, the mutant mice showed no reaction to doses as much as 100 times that administered to wild types. Expression analysis of Gb3/CD77 in mouse tissues with antibody revealed that low, but definite, levels of Gb3/CD77 were expressed in the microvascular endothelial cells of the brain cortex and pia mater and in renal tubular capillaries. Corresponding to the Gb3/CD77 expression, tissue damage with edema, congestion, and cytopathic changes was observed, indicating that Gb3/CD77 (and its derivatives) exclusively function as a receptor for VTs in vivo. The lethal kinetics were similar regardless of lipopolysaccharide elimination in VT preparation, suggesting that basal Gb3/CD77 levels are sufficient for lethal effects of VTs.

Widespread alteration of the genomic DNA is a hallmark of tumors, and alteration of genes involved in DNA maintenance have been shown to contribute to the tumorigenic process. The DNA polymerase of Saccharomyces cerevisiae is required for error-prone repair following DNA damage and consists of a complex between three proteins, scRev1, scRev3, and scRev7. Here we describe a candidate human homolog of S. cerevisiae Rev7 (hREV7), which was identified in a yeast two-hybrid screen using the human homolog of S. cerevisiae Rev3 (hREV3). The hREV7 gene product displays 23% identity and 53% similarity with scREV7, as well as 23% identity and 54% similarity with the human mitotic checkpoint protein hMAD2. hREV7 is located on human chromosome 1p36 in a region of high loss of heterozygosity in human tumors, although no alterations of hREV3 or hREV7 were found in primary human tumors or human tumor cell lines. The interaction domain between hREV3 and hREV7 was determined and suggests that hREV7 probably functions with hREV3 in the human DNA polymerase complex. In addition, we have identified an interaction between hREV7 and hMAD2 but not hMAD1. While overexpression of hREV7 does not lead to cell cycle arrest, we entertain the possibility that it may act as an adapter between DNA repair and the spindle assembly checkpoint. DNA damage is induced by a variety of endogenous and exogenous factors (1). Such DNA alterations include reactive oxygen damage, deamination, loss of nucleotides, nucleotide modifications, and DNA strand breaks. DNA damage repair has evolved to cope with these environmental and mutageninduced DNA alteration and plays a central role in maintaining the genetic stability of the organism (2, 3).Extensive studies of bacterial and yeast systems have identified components of the DNA repair machinery. In the yeast Saccharomyces cerevisiae, pyrimidine dimers induced by UV radiation damage are corrected by the RAD3 excision repair, the RAD6 postreplication repair, and the RAD52 recombinational repair pathways (for a review see Ref. 4). Interestingly, the removal of UV damage by the RAD6 pathway occurs by both error-free and error-prone (mutagenic) mechanisms (5, 6). The mutagenic repair of UV damage has been shown to require the UV revertible genes, REV1, REV3, and REV7, a lesion bypass polymerase complex consisting of a deoxycytidyl-transferase (Rev1), a polymerase catalytic subunit (Rev3), and a polymerase accessory protein (Rev7) (for review see Refs. 7 and 8). This polymerase complex has been termed polymerase .

Alterations of human chromosome 8p occur frequently in many tumors. We identified a 1.5-Mb common region of allelic loss on 8p22 by allelotype analysis. cDNA selection allowed isolation of several genes, including FEZ1. The predicted Fez1 protein contained a leucine-zipper region with similarity to the DNA-binding domain of the cAMPresponsive activating-transcription factor 5. RNA blot analysis revealed that FEZ1 gene expression was undetectable in more than 60% of epithelial tumors. Mutations were found in primary esophageal cancers and in a prostate cancer cell line. Transcript analysis from several FEZ1-expressing tumors revealed truncated mRNAs, including a frameshift. Alteration and inactivation of the FEZ1 gene may play a role in various human tumors.

The FEZ1͞LZTS1 gene maps to chromosome 8p22, a region that is frequently deleted in human tumors. Alterations in FEZ1͞LZTS1 expression have been observed in esophageal, breast, and prostate cancers. Here, we show that introduction of FEZ1͞LZTS1 into Fez1͞Lzts1-negative cancer cells results in suppression of tumorigenicity and reduced cell growth with accumulation of cells at late S-G 2͞M stage of the cell cycle. Fez1͞Lzts1 protein is hyperphosphorylated by cAMP-dependent kinase during cell-cycle progression. We found that Fez1͞Lzts1 is associated with microtubule components and interacts with p34 cdc2 at late S-G2͞M stage in vivo. Present data show that FEZ1͞LZTS1 inhibits cancer cell growth through regulation of mitosis, and that its alterations result in abnormal cell growth.

Background. Bone marrow metastasis often occurs in patients with neuroblastoma; therefore, a sensitive assay to detect occult neuroblastoma cells in bone marrow (BM) and peripheral blood (PB) is needed. The feasibility and clinical value of using the reverse transcriptase‐ (RT) polymerase chain reaction (PCR) to amplify mRNA for tyrosine hydroxylase (TH), the first enzyme of catecholamine synthesis, was evaluated to detect neuroblastoma cells in patient samples. Methods. Thirty‐eight patients with Stages I to IV neuroblastoma and eight healthy donors were included in this study. Bone marrow and PB samples obtained at diagnosis were examined for TH mRNA. After preparation of complementary DNA, the PCR was performed to amplify the TH gene. Results. Tyrosine hydroxylase mRNA was detected in neuroblastoma samples including a cell line and tumor tissues, but was not detected in normal BM or PB mononuclear cells. Neuroblastoma cells were detected at a level of 1 per 105‐6 normal PB mononuclear cells by this method. Tyrosine hydroxylase mRNA was detected in 18 of 38 BM samples, and all 12 BM samples with cytologic evidence of tumor cells were positive for TH mRNA by the RT‐PCR. Six of 26 patients without cytologic evidence of tumor cells in the BM were also positive for TH mRNA. TH mRNA was detected in BM samples from 1 of 14 patients with Stage I disease, 2 of 7 patients with Stage II disease, 1 of 3 patients with Stage III disease and all patients with Stage IV (11 patients) and IVS (3 patients) diseases. Tyrosine hydroxylase mRNA also was detected in 8 of 14 PB samples (one of five patients in Stages I, II or III and 7 of 9 in Stage IV or IVS). Conclusions. Reverse transcriptase‐polymerase chain reaction amplification of TH mRNA was a sensitive and specific method of detecting occult neuroblastoma cells in BM and PB samples. Neuroblastoma cells could be detected by this method in some BM samples that had no cytologic evidence of tumor cells and in some PB samples at the time of diagnosis. The clinical significance of these very low levels of neuroblastoma cells detected by RT‐PCR requires further investigation. Cancer 1995;75 2757–61.

Abstract-Smooth muscle cell (SMC) proliferation that results in neointima formation is implicated in the pathogenesis of atherosclerotic plaques and accounts for the high rates of restenosis that occur after percutaneous transluminal coronary angioplasty, a widespread treatment for coronary artery disease. Endothelial lesions trigger intense proliferative signals to the SMCs of the subintima, stimulating their reentry into the cell cycle from a resting G 0 state, resulting in neointima formation and vascular occlusion. Cellular proliferation is negatively controlled by growth-regulatory or tumorsuppressor genes, or both, such as the retinoblastoma gene family members (RB/p105, p107, RB2/p130). In the present study, we show that RB2/p130 inhibited SMC proliferation in vitro and in vivo. We used the rat carotid artery model of restenosis to demonstrate that adenovirus-mediated localized arterial transduction of RB2/p130 at the time of angioplasty significantly reduced neointimal hyperplasia and prevented restenosis. Furthermore, the ability of pRb2/p130 to block proliferation correlated with its ability to bind and sequester the E2F family of transcription factors, which are important mediators of cell cycle progression. These results imply that RB2/p130 could be an important target for vascular gene therapy. (Circ Res. 1999;85:1032-1039.)Key Words: restenosis Ⅲ adenovirus Ⅲ cell cycle Ⅲ pRb2 Ⅲ p130 Ⅲ gene therapy I t is well documented that chronic or acute injury to the arterial wall, such as that occurring with percutaneous transluminal coronary angioplasty (PTCA), induces the expression of a variety of growth factors and inflammatory cytokines that stimulate smooth muscle cell (SMC) proliferation and migration from the media into the intima, resulting in neointima formation and eventual restenosis. 1 Inhibition of neointima formation should greatly improve the effectiveness of PTCA in the long-term management of coronary artery disease. Numerous growth factors induce SMC proliferation through a variety of signal transduction pathways in vitro and in vivo. 2 This evidence suggests that critical regulatory proteins of the cell cycle machinery should be targeted instead of the upstream signal transduction molecules for effective cytostatic therapy of vascular proliferative disorders. [3][4][5] Various strategies have been used in animal models to prevent restenosis, including the transfer of the herpes simplex virus thymidine kinase associated with ganciclovir and the transduction of cell cycle regulatory genes such as RB/p105. 4,5 In particular, the retinoblastoma family proteins (pRb/ p105, p107, and pRb2/p130) are excellent candidates for vascular disease gene therapy. They are nuclear phosphoproteins with growth-suppressive properties that interact with specific members of the E2F transcription factor family (E2F-1 to E2F-5) and are regulated by phosphorylation/ dephosphorylation events in a cell cycle-dependent manner. 6,7 Previous studies show that the induction of pRb2/p130 expression growth arrests prolif...

These findings show that there is a substantial risk of tumor cell contamination in harvested PBSCs, although its incidence was lower than that in BM samples. We recommend that PBSCs would be better harvested during remission and should be examined for tumor contamination before use as a stem cell source.