CORRELATION OF HEAT-RESISTANCE AND HSP-70A MESSENGER-RNA LEVELS IN HUMAN TUMOR-CELLS MEASURED BY COMPETITIVE QUANTITATIVE POLYMERASE CHAIN-REACTION

Purpose: Several HSP-70 genes have been cloned and sequenced in human cells. Among these genes, the HSP-70A mRNA and protein levels correlat e best with the development and decay of thermotolerance and intrinsic thermal sensitivity. Leukemic and nonleukemic human tumor cells expre ss low levels of the normally heat inducible HSP-70A mRNA in control n onheated cells. Using a competitive quantitative polymerase chain reac tion, we have measured the levels of mRNA for this gene and have corre lated it with both transient and intrinsic thermal sensitivity of tumo r cells. Such studies were also extended to tumor samples obtained fro m patients. Methods and Materials: In these studies, the plasmid phHSP -70 which contains the entire human HSP-70A gene was modified by the i nsertion of the T7 promoter at the 5'-end untranslated region as well as the insertion of a 23 bp synthetic linker at the BamH1 site in the promoter region of the HSP-70A gene. The PCR primers were located such that the amplified fragment contained the linker. Using the T7 polyme rase, the HSP-70A mRNA was transcribed from this plasmid (phHSP-70L) i n vitro. A known amount of HSP-70A mRNA was then added to the total RN A prepared from the cell samples or from the tumor tissues obtained fr om patients. Using the components of the PCR reaction plus known amoun ts of HSP-70A mRNA synthesized from phHSP-70L and unknown amounts of t otal cellular RNA, the samples were amplified and analysed on a denatu ring acrylamide gel. The PCR products obtained from phHSP-70L were 23 bp larger than the PCR products obtained from the cell samples due to the addition of the synthetic linker to the HSP-70A gene in phHSP-70L and therefore, the two products could be easily distinguished from eac h other and quantitated. The alpha-P-31-dCTP incorporated in each samp le was quantitated by AMBIS Scanner. When the P-32-counts were equal i n the known and the unknown samples, the amount of the HSP-70A mRNA wa s taken to be equal in the known and the unknown sample. Results: The results show that HSP-70A mRNA levels can be used to predict the survi val levels during the development and decay of thermotolerance. In non leukemic human tumor cell lines, there are as much as 40-50-fold induc tion of HSP-70A mRNA levels during the peak of thermotolerance. In leu kemic cell lines, however, HSP-70A mRNA levels are induced only by thr ee-fold during the same time period. These differences between the lev els of HSP-70A mRNA positively correlate with the amount of tolerance development in leukemic and nonleukemic tumor cells. HSP-70A mRNA leve ls also vary in different tumor cells under nonheated conditions and t here is a positive correlation between HSP-70A mRNA levels in nonleuke mic human tumor cells and the level of their intrinsic thermal resista nce. Conclusion: HSP-70A mRNA levels can be used to predict the intrin sic thermal sensitivity of nonleukemic human tumor cells.