The HspA2 protein localizes in nucleoli and centrosomes of heat shocked cancer cells

Abstract

The human HSPA2 gene, which belongs to the HSP70 family of heat shock genes, is a counterpart of rodent testis-specific HspA2 gene. Rodent genes are expressed mainly in pachytene spermatocytes, while transcripts of human HSPA2 gene have been detected in various normal somatic tissues, albeit translation of the messenger RNA into corresponding protein has not been yet unambiguously demonstrated, except for several cancer cell lines. The aim of our work, a first step in search for HspA2 function in cancer cells, was to establish its intracellular localization at physiological temperature and during heat shock. First, we used qRT-PCR and a highly specific antibody to select cell lines with the highest expression of the HspA2 protein, which turned out to be A549 and NCI-H1299 lines originating from non-small cell lung carcinoma (NSCLC). Significant expression of the HspA2 was also detected by immunohistochemistry in primary NSCLC specimens. Intracellular localization of the HspA2 was studied using both the specific anti-HspA2 polyclonal antibody and transfection of cells with fusion proteins HspA2-EGFP and mRFP-HspA2. We found that, at physiological temperature, the HspA2 was localized primarily in cytoplasm whereas, during heat shock, localization shifted to nucleus and nucleoli. Moreover, we demonstrate that in heat-shocked cells HspA2 accumulated in centrosomes. Our results suggest that the HspA2, like Hsp70 protein, can be involved in protecting nucleoli and centrosomes integrity in cancer cells subjected to heat shock and, possibly, other cellular stressors.

The expression pattern of the 70-kDa heat shock protein Hspa2 in mouse tissues

“…However, anti-Hspa2 antibody used in this work was highly speciWc and showed no cross-reactivity with those Hsp70 proteins ( Fig. 1a; also Rosario et al 1992;Scieglinska et al 2008). Moreover, no unspeciWc binding of the antibody to proteins extracted from mouse organs was detected (Fig.…”

“…However, in many others somatic tissues Hspa2 transcription was shown to be very low in rodents (Scieglinska et al 1997), but abundant in human (Bonnycastle et al 1994;Roux et al 1994). In addition, the expression of HSPA2 was also reported in several human cancer cell lines and primary cancers (Rohde et al 2005;Piglowski et al 2007;Scieglinska et al 2008).…”

“…Anti-Hspa2 rabbit polyclonal antibody was generated using the NH2-SKLYQGGPGGGGSSGGPT peptide corresponding to amino acids 611-628 in the mouse Hspa2 sequence (Rosario et al 1992;Scieglinska et al 2008). For immunization the peptide was C-terminally-conjugated with bovine thyroglobulin (Sigma-Genosys Custom Peptide Antisera Facility).…”

“…Here, we aimed to analyze the expression pattern of Hspa2 protein outside testes of adult mice taking advantage of antibody raised against synthetic peptide from C-terminal domain that speciWcally diVers Hspa2 from other Hsp70s (Rosario et al 1992;Scieglinska et al 2008). Additionally, we have compared distribution of Hspa2 protein with activity of Hspa2 promoter studied in Hspa2-EGFP transgenic mice.…”

Recent progress in histochemistry and cell biology

“…With regard to the HSPA family members HSPA1 [stress-inducible chaperone found to be expressed in primary tumors, but also with stress-independent expression in certain normal epithelial cells (Mangurten et al 1997;Trautinger et al 1995)] and HSPA2 [the expression of which was originally ascribed exclusively to certain testicular cell types (Huszar et al 2000;Son et al 1999), suggesting a role for HSPA2 in spermatid development/progression of spermatogenesis (Dix et al 1997;Govin et al 2006;Huszar et al 2000); however, an additional role in tumorigenesis has also been suggested (Piglowski et al 2007;Rohde et al 2005;Scieglinska et al 2008)] expression pattern have rarely been investigated in normal, non-stressed/pathological human tissues. Therefore, Scieglinska et al (2011) performed a thorough tissue-microarray based immunohistochemical study to collect new extensive data on the possible constitutive cell type-specific expression of the HSPA family members HSPA1 and HSPA2 in human somatic tissues, screening samples of 24 normal tissues derived from up to 11 donors.…”