Across a wide range of species from E. coli
to humans, HSP70 is the most highly conserved HSP at the sequence level and displays the largest, most consistent increase in expression following stress (22
). Increased levels of HSP70 protein are cytoprotective (33
). Cells subjected to a nonlethal heat shock increase cellular HSP70 levels, and those levels correlate with a transient resistance to higher, normally lethal temperatures (29
). A review of results suggests that inactivation of either of the two genes (Hsp70.1
) results in deficient maintenance of acquired thermotolerance and increased sensitivity to heat stress-induced apoptosis (8
). The synthetic or protein rescue functions of HSP70 are counterbalanced by participation in the ubiquitin pathway that clears the cell of unstable or damaged proteins by proteosome degradation (2
). Because of such functions, HSP70 also influences cell death and the cell transformation process (53
). Consistent with such a function of HSP70, we found that Hsp70.1/3
-null mice are lighter in weight and have elevated levels of spontaneous genomic instability. Two possible and not mutually exclusive reasons for the lighter weight of Hsp70.1/3−/−
mice could be the loss of cells, increased population doubling time, and/or both. Results described for Fig. support the argument that Hsp70.1/3 affect the cell growth as Hsp70.1/3−/−
MEFs have a longer doubling time than Hsp70.1/3+/+
MEFs do. The difference in population doubling cannot be attributed to the cell cycle differences, as no major difference in the distribution of cells in different phases of the cell cycle among cells with and without Hsp70.1/3 was found (data not shown). One possible mechanism contributing to the increase in population doubling could be genomic instability.
Both in vivo and in vitro studies support the argument about the role of Hsp70.1/3 in genomic stability. It has been reported elsewhere that HSP70 interacts with human apurinic/apyrimidinic endonucleases and enhances the specific endonuclease activity of HAP1 (24
), thus supporting the idea that Hsp70.1/3 play a role in the repair of DNA damage. Hsp70.1/3−/−
mice have a higher ratio of normochromatic to polychromatic erythrocytes than do Hsp70.1/3+/+
mice. Furthermore, Hsp70.1/3−/−
mice also have a higher frequency of micronuclei in bone marrow erythrocytes than do Hsp70.1/3+/+
mice. Both the ratio of normochromatic to polychromatic erythrocytes and the frequency of micronuclei in erythrocytes significantly increased after heat shock treatment in Hsp70.1/3−/−
mice compared with Hsp70.1/3+/+
mice. In addition, genomic instability was also found in germ cells of Hsp70.1/3−/−
mice. Heat shock enhanced significantly the frequency of aberrant spermatocytes in Hsp70.1/3−/−
mice compared with Hsp70.1/3+/+
mice. These results are consistent with the effects of heat on testicular weight loss and spermatogenesis (15
) with Hsp70.1/3 deficiency enhancing heat-mediated genomic instability.
The in vivo results are consistent with the in vitro studies of chromosome damage repair analysis after heat or heat and IR treatment, supporting the argument that Hsp70.1/3 play a role combating spontaneous or heat-induced genotoxic stress. Based on the fact that heat treatment did enhance significantly the frequency of micronuclei in Hsp70.13−/− mice compared to that in Hsp70.1/3+/+ mice, in vivo studies are thus in agreement with the role of HSP70 in repair of DNA damage. The role of HSP70 in DNA damage repair is further strengthened by the fact that MEFs from Hsp70.1/3−/− mice have a higher frequency of spontaneous as well as heat-modulated and IR-induced chromosome aberrations than do Hsp70.1/3+/+ cells.
Inactivation of Hsp70.1/3 does lead to enhanced heat-modulated IR-induced cell killing, and the enhanced cell killing correlates with higher S-phase-specific chromosome residual damage. Further, a role for Hsp70.1/3 in S phase is evident from the fact that deficient cells demonstrate radioresistant DNA synthesis after IR treatment. Interestingly, expression of Hsp70.1 in Hsp70.1/3−/− cells rescued the radioresistant DNA synthesis phenotype in such cells, thus supporting the role of Hsp70.1/3 in the IR response in S phase.
HSP70 family members transiently associate with key molecules of the cell cycle control systems, including p53, Cdk4, pRb, p27/Kip1, cMyc, Wee-1, and some others, which affect cell growth (7
). Cell growth is also affected by several other factors e.g., defective telomere metabolism (35
). HSP70 is known to interact with TERT, which is involved in telomere metabolism (10
). There is recent evidence that telomerase may have functions other than the synthesis of telomeric repeats of the G strand (47
). Ectopic expression of TERT prevents replicative senescence in several cell types including fibroblasts and epithelial cells (3
). It may also exert an antiapoptotic action at an early stage of the cell death process prior to mitochondrial dysfunction and caspase activation (12
). It has been proposed previously that telomere shortening during human replicative aging generates antiproliferative signals which mediate p53-dependent G1
arrest as observed in senescent cells (50
). In support of this idea, Wong et al. (51
) reported that telomere dysfunction in mTerc-null mice impairs DNA repair and subsequently leads to cell growth arrest. Goytisolo et al. (14
) reported radiosensitivity of the late-generation telomerase-KO mice. Choi et al. (6
) demonstrated that telomerase expression suppresses senescence-associated genes in Werner syndrome cells. Sharma et al. (47
) reported that hTERT interacts with the telomeres, influences the interaction of telomeres with the nuclear matrix, and leads to transcriptional alteration along with increased genomic stability and enhanced DNA repair. Thus, some of the effects of TERT and HSP70 seem to be similar. Present results clearly demonstrate that the inactivation of Hsp70.1/3 does influence telomerase activity, as Hsp70.1/3−/−
cells have 2.5-fold-less telomerase activity than do Hsp70.1/3+/+
cells. Cells deficient in Hsp70.1/3 also showed loss of telomeric signals as well as chromosome end associations, which are known to contribute to the genomic instability.
In addition to HSP70's unique function in protecting the cells from stress-related damage, HSP70s have attracted attention in the cancer field by their aberrant expression in most human tumors in general and physically interacting with cellular proteins of vital biological importance including tumor suppressors like p53 (7
). Although it is well established that tumor cells have a higher expression of HSP70, the present results suggest that the lack of such expression leads to genomic instability and higher IR-induced cell killing, both phenomena which are linked with oncogenic transformation. Consistent with such a hypothesis, Hsp70.1/3−/−
cells have a higher frequency of oncogenic transformation, suggesting that the absence of such gene products is essential to suppress tumor formation. Interestingly, the results presented here suggest a correlation between the negative effects of Hsp70.1/3 on reduced telomerase activity with telomere instability and reduced growth potential as well as increased radiosensitivity. While it is likely that these different effects are the result of inactivation of Hsp70.1/3 and, therefore, of independent origin, it remains possible that interference with DNA repair and telomere functions could contribute to the overall growth defects. The chromosomal end-to-end associations with telomeric sequences at the fusion points could reflect an inhibition of the TRF2 protein, and the resulting end-to-end association of chromosomes may induce cell cycle arrest and genomic instability. Therefore, we suggest that the overall growth phenotypes and radiosensitivity observed in Hsp70.1/3
-null cells may be the result of a combination of effects. Thus, our results show that inactivation of Hsp70.1/3 influences cell growth as well as cell survival after IR treatment, telomere stability, chromosome repair, and oncogenic transformation. These observations are consistent with a model that predicts that Hsp70.1/3 have a critical role in stress response. We therefore propose that Hsp70.1/3 can play a critical role during the process of oncogenesis. Further experiments are required to determine the specific contributions of Hsp70.1/3 in the DNA damage repair process.