Use of polymerase chain reaction to detect the expression of the Mr 70,000 heat shock genes in control or heat shock leukemic cells as correlated to their heat response.
The expression of the Mr 70,000 heat shock protein (HSP-70) in heat-resistant variants or heat-shocked cells has been correlated with development of thermal resistance. In these studies polymerase chain reaction (PCR) was used to detect low levels of HSP-70 mRNA present in control, unheated cells to investigate the possibility of predicting the intrinsic heat response in various leukemic cells. The expression of two human heat shock genes in control or heat-shocked cells was investigated. Synthetic primers and probes from the untranslated region of the two HSP-70 genes sequenced by Hunt and Morimoto (HSP-70A)(C. Hunt and R. I. Morimoto, Proc. Natl. Acad. Sci. USA, 82: 6455-6459, 1985) and Voellmy et al. (HSP-70B)(R. Voellmy et al., Proc. Natl. Acad. Sci. USA, 82: 4949-4953, 1985) were used in PCR reactions to follow expression in control or heat-shocked leukemic K562, KG-1, and HL-60 cells. The PCR results were correlated with heat response and patterns of protein synthesis in these cells. Results indicate that, among leukemic cells, K562 was much more resistant to killing by heat shock than either KG-1 or HL-60 cells. All control cells, however, expressed the HSP-70B gene. Of the three leukemic cells tested, K562 was the most heat resistant and constitutively expressed the HSP-70A mRNA and the heat-inducible HSP-70 protein. KG-1 and HL-60 cells did not express this gene in unheated cells. All heat-shocked cells expressed the HSP-70A mRNA and the heat-inducible HSP-70 protein. However, there was no significant increase in the mRNA level of the HSP-70B in heat-shocked leukemic cells as measured by PCR or the S1-nuclease protection assay. Other cells including normal human bone marrow and normal and tumorous tissues of the colon and breast all expressed both genes in control cells. Normal breast tissue expressed less mRNA for HSP-70B gene than the tumor tissue obtained from the same patient. In all studies the amplified beta-actin mRNA expression was used as an internal standard. These studies indicate that HSP-70B gene is expressed in all control leukemic cells. The expression of this gene did not seem to correlate with intrinsic heat resistance. The HSP-70A expression correlated with intrinsic and transient heat resistance. These studies also indicate that both HSP-70 genes in humans may be expressed in a variety of unheated normal and tumorous tissues more so than previously reported.
Mivechi
N F
NF
Department of Radiation Research and Molecular Genetics, City of Hope National Medical Center, Duarte, California.
Rossi
J J
JJ
D001483:Base Sequence; D001853:Bone Marrow / Q000502:physiology; D015180:Electrophoresis, Gel, Two-Dimensional; D005784:Gene Amplification; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000235:genetics*; D006358:Hot Temperature; D006801:Humans; D066298:In Vitro Techniques; D007938:Leukemia / Q000235:genetics; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D006360:Heat-Shock Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D015719:Single-Strand Specific DNA and RNA Endonucleases