We recently isolated human cDNA fragments that render MCF-7 breast cancer cells resistant to cell death caused by Pseudomonas exotoxin, Pseudomonas exotoxinderived immunotoxins, diphtheria toxin, and tumor necrosis factor. We report here that one of these fragments is We have used expression cloning to isolate cDNAs containing plasmids that cause MCF-7 breast cancer cells to become resistant to immunotoxins (1). These plasmids also render the cells resistant to native Pseudomonas exotoxin (PE) and diphtheria toxin (DT); thus the resistance is due to the action of the toxin moiety of immunotoxins and not due to reduced expression of the antigen to which the immunotoxin is directed. One cDNA plasmid (p17 in ref. 1) was particularly interesting because the phenotype it produced could not be attributed to known mechanisms of PE and DT resistance (2, 3). In fact, when cells transfected with this plasmid were exposed to toxin, modification of elongation factor EF2 and inhibition of protein synthesis was comparable to toxin-sensitive controls, yet the cells did not die. Thus, this cDNA appears to be involved in a mechanism affecting the sensitivity of cells to toxin after the primary action of the toxin, inhibition of protein synthesis, has occurred. The observation that PE and DT can induce apoptosis (4-6) and the observation that this plasmid also confers resistance to tumor necrosis factor (TNF) a and ,B indicate that this cDNA could mediate resistance by interference with apoptosis (unpublished results). Here we describe that the isolated resistance plasmid that mediates toxin as well as TNF resistance to MCF-7 cells contains an antisense cDNA fragment homologous to the yeast CSE1 gene (7). We report the sequence of the human CSE1 homologue and present evidence that this gene may play a role in cell proliferation.t MATERIALS AND METHODSExpression Cloning and Immunotoxin Selection of cDNA Plasmids. Human cDNAs that confer resistance to the immunotoxin B3(Fv)-PE38KDEL were isolated by expression cloning and immunotoxin selection from a cDNA library in pCDM8 (HeLa cDNA expressed from a cytomegalovirus promoter followed by simian virus 40 poly(A) as described (1). B3(Fv)-PE38KDEL is a fusion protein composed of a truncated form of PE and the Fv region of monoclonal antibody B3 that binds to a carbohydrate present on many carcinomas and cancer cell lines-e.g., on MCF-7 cells-and kills such cells (8). pCDM/HE17 (p17 in ref. 1) is a plasmid that confers resistance not only to immunotoxin, PE, and DT but also to TNF.

The CAS (cellular apoptosis susceptibility) gene is the human homolog of the yeast chromosome segregation gene CSE1. CAS may have a dual function in mammalian cells, one in apoptosis and another in cell proliferation. We have now mapped the CAS gene to chromosome 20q13. This region is known to harbor amplifications that correlate with aggressive breast cancer. Southern hybridizations with a CAS cDNA fragment and fluorescent in situ hybridization (FISH) with a P1 clone containing the CAS gene show elevated copy numbers in one leukemia, three of four colon, and in three of seven breast cancer cell lines. Elevated CAS copy number in CEM leukemia and COLO201 colon cancer cells was attributable to additional copies of chromosome 20. In SW480 and COLO205 colon cancer cells CAS is part of aberrant chromosomes containing large parts of 20q. In breast cancer cells CAS is also part of aberrant 20q chromosomes (MDA-MB-157 and UACC-812) or of additional 20q isochromosome in MDA-MB-134. In MDA-MB361 and BT-474 breast cancer cells CAS is separated from other markers centromeric and telomeric of CAS on 20q. MDA-MB 361 contains one additional copy of CAS, separated from the centromeric 20q control probe. BT-474 cells have up to 12 additional CAS copies that we separated from nearby telomeric and centromeric probes on 20q and that are translocated to abnormal chromosomes.

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