This study has demonstrated that selective depletion of tNASP from PC-3, HeLa and PWR-1E cell lines in culture leaves sNASP unaffected and results in distinctly different effects. Using lentiviral expression of tNASP shRNA, all three cell lines had decreased HSPA2 protein levels, yet only PC-3 and HeLa cells increased expression of p21 and entered apoptosis. NASP has been detected in all dividing cells, but only cancer, transformed, germ, and embryonic cells have a high level of expression of the tNASP splice variant . Depletion of both tNASP and sNASP has been shown to be embryonic lethal and without sufficient NASP HeLa and U2OS cells were unable to enter S phase . However, these studies did not distinguish whether tNASP and sNASP played separate roles and raised the question why rapidly dividing cells such as cancer or embryonic cells expressed tNASP while adult somatic cells did not.
The results reported in this study clearly demonstrated that in prostate cancer PC-3 cells and cervical cancer HeLa cells depletion of tNASP caused significant anti-proliferative effects and induced apoptosis while the non-tumorigenic prostate epithelial PWR-1E cells did not experience these changes in the time span observed. Cells depleted of tNASP slowed down their proliferation and were blocked at the G1/S border of the cell cycle, which is consistent with our earlier report .
The activation of p53 could result in apoptosis, cell-cycle arrest, DNA repair or senescence [38, 39]; however activation of p53 is not relevant in our study because we used cell lines with non-functional, inhibited or mutated p53. HeLa cells, which originate from adenocarcinoma of the cervix, contain wild-type p53 alleles together with the DNA of specific types of human papillomaviruses  and the transcriptional stimulatory activity of HeLa cell p53 is strongly repressed by one of the oncogenic papilomavirus products, E6 protein . In PC-3 prostate cancer cells p53 is absent due to deletion of a base pair in the single copy gene and that generates a frame shift and a new immediate stop codon [42, 43]. PWR-1E cells were immortalized by adenovirus 12-SV40 and although they show strong nuclear staining for p53  its activity is inhibited by the binding between p53 and SV40 Large T-antigen . In this study p53 protein could only be detected on Western blots in PWR-1E cells (Figure 2).
The up regulation of p21 appears to be important in the cell lines we studied. Expression of p21 mRNA increased in all three cell lines; however protein levels were increased only in PC-3 and HeLa cells. p21 is known to be a potent cell cycle progression inhibitor because it inhibits the activity of cyclin-dependant kinase-cyclins A, D, and E complexes . Although it is well established that p21 mediates p53-dependant G1/S phase growth arrest in response to DNA damage [46, 47], p21 expression could be induced by mechanisms independent of p53. In this case the expression of p21 is up regulated by different transcription factors: E2F1 through the activation of Ras , SP1, SP3, AP2, HOXA10, STAT , AP-1 .
Because tNASP is a chaperone for linker histones and transports them into the nucleus in an energy and nuclear localization signal-dependent manner , the depletion of tNASP implies that there could be a simultaneous depletion of H1 histones in the nucleus. Depletion of linker histones has been reported to be sufficient for altering the expression of subsets of genes, the up regulation of p21 in particular, and for G1-phase cell cycle arrest in human breast cancer cells . Regulation of DNA replication through multiple pathways in egg extracts has been reported to be dependent on sufficient linker histones . Decreased levels of H1 histones down to 50% from their normal level caused profound chromatin structural changes, including global nucleosome spacing, reduced chromatin compaction, and decreases in core histone modifications . Moreover, linker histones are critical for the interaction of tNASP and HSPA2. We previously reported  that in mouse spermatogenic cells tNASP in the presence of CDC2 requires H1 linker histones to significantly increase HSPA2 ATPase activity. Therefore, depletion of tNASP should result in lesser amounts of HSPA2 activity implying that the interaction of tNASP and HSPA2 may be an important factor in the differential response of cancer cell lines to the depletion of tNASP. All three cell types in this study showed a decrease in HSPA2 protein levels (Figure 2) and the ATPase activity of HSPA2 was significantly increased in the presence of tNASP, but not sNASP (Figure 7).
HSPA2 activity was previously reported as a necessary chaperone for formation of an active CDC2-cyclin B1 complex in pachytene spermatocytes as a condition for successful completion of the G2/M transition . HSPA2 is not only essential for spermatogenesis , but is required for tumor cell growth and survival . HSPA2 depletion resulted in a senescence-like condition, arrest in the G1 phase of the cell cycle and up or down regulation of genes such as MIC-1 and p21 . Interestingly HSPA2 is required for survival of cancer cells , while non-tumorigenic cells (mammary MCF-10A, HBL-100, and prostate PNT1A) displayed no changes in survival after depletion of HSPA2. This selective effect is similar to our observation that although PWR-1E cells lost HSPA2 protein their survival was not affected.