Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide.
We have used antisense RNA technology to inhibit endogenous parathyroid hormone-related peptide (PTHRP) production in the established human keratinocyte cell line, HPK1A, in order to assess the role of PTHRP as a potential modulator of cell growth. Rat PTHRP cDNA was cloned into the replication defective retroviral vector pYN in an antisense orientation and a stable cell line (HPK1A-AS) was generated after infection by amphotropic virus and selection by the neomycin derivative, G418. Expression of the transfected antisense sequence was confirmed with an RNA sense probe for PTHRP. The effect of the retrovirally mediated gene transfer on the endogenous PTHRP transcript was examined with an RNA antisense probe which demonstrated an absence of the endogenous transcript in HPK1A-AS cells. A 1.6-kilobase transcript was, however, present in equivalent quantities in both uninfected HPK1A and pYN-infected (HPK1A-pYN) cells. Immunocytochemistry and assessment of PTHRP secretion into the medium using an NH2-terminal radioimmunoassay and a UMR 106 adenylate cyclase bioassay confirmed the absence of PTHRP in HPK1A-AS cells. Examination of the inhibition of PTHRP production on cell growth demonstrated a reduction in doubling time and an increase in [3H]thymidine incorporation. Cell cycle analysis showed an increase in the proportion of the cell population in the S phase (relative to G0/G1) in HPK1A-AS cells compared to HPK1A or HPK1A-pYN cells. These data, therefore, indicate that endogenous PTHRP acts as an effective inhibitor of cell growth in this keratinocyte model and that this action occurs, at least in part, by diminishing entry into the S phase of the cell cycle. Furthermore, the antisense RNA method is a potent one to evaluate the cellular actions of PTHRP.
Kaiser
S M
SM
Department of Medicine, McGill University, Montreal, Canada.
Laneuville
P
P
Bernier
S M
SM
Rhim
J S
JS
Kremer
R
R
Goltzman
D
D
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002453:Cell Cycle; D002455:Cell Division / Q000235:genetics*; D002460:Cell Line; D004247:DNA; D006801:Humans; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D008969:Molecular Sequence Data; D044162:Parathyroid Hormone-Related Protein; D010957:Plasmids; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D016372:RNA, Antisense; D011863:Radioimmunoassay; D051381:Rats; D013379:Substrate Specificity; D013936:Thymidine / Q000378:metabolism
C470121:PTHLH protein, human; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D016372:RNA, Antisense; D004247:DNA; D013936:Thymidine