Genetic identity and differential expression of p38.5 (Haymaker) in human malignant and nonmalignant cells.
Previous studies from our laboratory revealed a novel protein of 38.5 kD on the surface of malignant cell lines of hematopoetic origin that exhibit susceptibility to naive natural killer (NK) cell-mediated lysis. In contrast, p38.5 was not detected on the surface of NK cell-resistant carcinoma cell lines or normal cells. We now report that this protein is differentially expressed, intracellularly, in malignant cell lines of both hematopoetic and epithelial origin compared with nonmalignant cells. To characterize p38.5 further, we used a previously developed antipeptide antibody (anti-11-mer) to probe cDNA expression libraries and subsequently performed 5' extension by rapid amplification of cDNA ends (RACE). Sequence analyses of these cDNA clones reveal open reading frames (ORFs) that include the previously identified 11-mer peptide from purified, native p38.5 and that have identical sequences to a gene of unknown function on chromosome 19. Nucleotide sequence data obtained from these cDNA clones, as well as analysis of the genomic sequence, permitted design of primers for reverse transcriptase-polymerase chain reaction (RT-PCR) that resulted in a cDNA clone encoding an ORF of 361 amino acids; the clone was identical to a sequence encoded by an unpublished mRNA in GenBank. Anti-p38.5 antibody against the 11-mer peptide encoded in exon 5 and against a 25-mer peptide encoded in exon 1 both reacted with the same protein in immunoprecipitation studies, providing further evidence of identity. RT-PCR and Northern blot analyses both demonstrated p38.5 gene transcripts in normal cells, nonmalignant cell lines and malignant cell lines of epithelial as well as hematopoietic origin. Semiquantitative studies revealed a greater level of p38.5 gene transcription in malignant cell lines compared with nonmalignant cells. Immunoblot analyses of protein expression confirmed and extended the latter studies by revealing substantially greater levels of the 38.5 kD protein in whole cell extracts of malignant cell lines compared with nonmalignant cells. Quantitative differences in detection of the 38.5 kD protein and mRNA in NK susceptible- hematopoietic malignancies compared with NK resistant-carcinomas were not observed. These experiments suggest that the p38.5 gene (Haymaker) is widely expressed in human cells of different tissue origins but that elevated expression is associated with the malignant phenotype.
Das
B
B
Department of Surgery, State University of New York Downstate Medical Center, Brooklyn, NY 11203, USA.
Tao
S Z
SZ
Mushnitsky
R
R
Norin
A J
AJ
D000595:Amino Acid Sequence; D003429:Cross Reactions; D006801:Humans; D015151:Immunoblotting; D007694:Killer Cells, Natural / Q000276:immunology*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D009369:Neoplasms / Q000276:immunology / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D011956:Receptors, Cell Surface / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C106253:LOC133728 protein, human; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface