Various Anti-HSPA2 Antibodies Yield Different Results in Studies on Cancer-Related Functions of Heat Shock Protein A2.

Heat shock proteins (HSPs) constitute a major part of the molecular chaperone system and play a fundamental role in cell proteostasis. The HSPA (HSP70) family groups twelve highly homologous HSPA proteins. Certain HSPAs are regarded as important cancer-related proteins, prospective therapeutic targets for cancer treatment, and also as potential cancer biomarkers. Heat Shock Protein A2 (HSPA2), a testis-enriched chaperone and one of the least characterized members of the HSPA family, has recently emerged as an important cancer-relevant protein with potential biomarker significance. Nevertheless, conflicting conclusions have been recently drawn both according to HSPA2 role in cancer cells, as well as to its prognostic value. In this work we have shown that one of the serious limitations in HSPA2 protein research is cross-reactivity of antibodies marketed as specific for HSPA2 with one or more other HSPA(s). Among non-specific antibodies were also those recently used for HSPA2 detection in functional and biomarker studies. We showed how using non-specific antibodies can generate misleading conclusions on HSPA2 expression in non-stressed cancer cells and tumors, as well as in cancer cells exposed to proteotoxic stress. Our findings addressed concerns on some published studies dealing with HSPA2 as a cancer-related protein.

Novel role for the testis-enriched HSPA2 protein in regulating epidermal keratinocyte differentiation.

HSPA2, a poorly characterized member of the HSPA (HSP70) chaperone family, is a testis-enriched protein involved in male germ cell differentiation. Previously, we revealed that HSPA2 is present in human stratified epithelia, including epidermis, however the contribution of this protein to epithelial biology remained unknown. Here, we show for the first time that HSPA2 is expressed in basal epidermal keratinocytes, albeit not in keratinocytes exhibiting features attributed to primitive undifferentiated progenitors, and participates in the keratinocyte differentiation process. We found that HSPA2 is dispensable for protection of HaCaT keratinocytes against heat shock-induced cytotoxicity. We also shown that lentiviral-mediated shRNA silencing of HSPA2 expression in HaCaT cells caused a set of phenotypic changes characteristic for keratinocytes committed to terminal differentiation such as reduced clonogenic potential, impaired adhesiveness and increased basal and confluency-induced expression of differentiation markers. Moreover, the fraction of undifferentiated cells that rapidly adhered to collagen IV was less numerous in HSPA2-deficient cells than in the control. In a 3D reconstructed human epidermis model, HSPA2 deficiency resulted in accelerated development of a filaggrin-positive layer. Collectively, our results clearly show a link between HSPA2 expression and maintenance of keratinocytes in an undifferentiated state in the basal layer of the epidermis. It seems that HSPA2 could retain keratinocytes from premature entry into the terminal differentiation process. Overall, HSPA2 appears to be necessary for controlling development of properly stratified epidermis and thus for maintenance of skin homeostasis.

Cell type-dependent modulation of the gene encoding heat shock protein HSPA2 by hypoxia-inducible factor HIF-1: Down-regulation in keratinocytes and up-regulation in HeLa cells.

HSPA2 belongs to the multigene HSPA family, whose members encode chaperone proteins. Although expression and function of HSPA2 is mainly associated with spermatogenesis, recent studies demonstrated that in humans, the gene is active in various cancers, as well as in normal tissues, albeit in a cell type-specific manner. In the epidermis, HSPA2 is expressed in keratinocytes in the basal layer. Currently, the mechanisms underlying the regulation of HSPA2 expression remain unknown. This study was aimed at determining whether HIF-1 and its binding site, the hypoxia-response element (HRE) located in the HSPA2 promoter, are involved in HSPA2 regulation. As a model system, we used an immortal human keratinocyte line (HaCaT) and cervical cancer cells (HeLa) grown under control or hypoxic conditions. Using an in vitro gene reporter assay, we demonstrated that in keratinocytes HSPA2 promoter activity is reduced under conditions that facilitate stabilization of HIF-1α, whereas HIF-1 inhibitors abrogated the suppressive effect of hypoxia on promoter activity. Chromatin immunoprecipitation revealed that HIF-1α binds to the HSPA2 promoter. In keratinocytes, hypoxia or overexpression of a stable form of HIF-1α attenuated the expression of endogenous HSPA2, whereas targeted repression of HIF-1α by RNAi increased transcription of HSPA2 under hypoxia. Conversely, in HeLa cells, HSPA2 expression increased under conditions that stimulated HIF-1α activity, whereas inhibition of HIF-1α abrogated hypoxia-induced up-regulation of HSPA2 expression. Taken together, our results demonstrate that HIF-1 can exert differential, cell context-dependent regulatory control of the HSPA2 gene. Additionally, we also showed that HSPA2 expression can be stimulated during hypoxia/reoxygenation stress.

Synthetic genistein glycosides inhibiting EGFR phosphorylation enhance the effect of radiation in HCT 116 colon cancer cells.

The need to find new EGFR inhibitors for use in combination with radiotherapy in the treatment of solid tumors has drawn our attention to compounds derived from genistein, a natural isoflavonoid. The antiproliferative potential of synthetic genistein derivatives used alone or in combination with ionizing radiation was evaluated in cancer cell lines using clonogenic assay. EGFR phosphorylation was assessed with western blotting. Genistein derivatives inhibited clonogenic growth of HCT 116 cancer cells additively or synergistically when used in combination with ionizing radiation, and decreased EGFR activation. Our preclinical evaluation of genistein-derived EGFR inhibitors suggests that these compounds are much more potent sensitizers of cells to radiation than the parent isoflavonoid, genistein and indicate that these compounds may be useful in the treatment of colon cancer with radiation therapy.

HSPA2 overexpression protects V79 fibroblasts against bortezomib-induced apoptosis.

Human HSPA2 is a member of the HSPA (HSP70) family of heat-shock proteins, encoded by the gene originally described as testis-specific. Recently, it has been reported that HSPA2 can be also expressed in human somatic tissues in a cell-type specific manner. The aim of the present study was to find out whether HSPA2 can increase the resistance of somatic cells to the toxic effect of heat shock, proteasome inhibitors, and several anticancer cytostatics. We used a Chinese hamster fibroblast V79 cell line because these cells do not express the HSPA2 and cytoprotective HSPA1 proteins under normal culture conditions and show limited ability to express HSPA1 in response to heat shock and proteasome inhibitors. We established, by retroviral gene transfer, a stable V79/HSPA2 cell line, which constitutively overexpressed HSPA2 protein. The major observation of our study was that HSPA2 increased long-term survival of cells subjected to heat shock and proteasome inhibitors. We found, that HSPA2 confers resistance to bortezomib-induced apoptosis. Thus, we showed for the first time that in somatic cells HSPA2 can be a part of a system protecting cells against cytotoxic stimuli inducing proteotoxic stress.

Aneugenic effects of the genistein glycosidic derivative substituted at C7 with the unsaturated disaccharide.

Genistein, due to its recognized chemopreventive and antitumour potential, is a molecule of interest as a lead compound in drug design. Recently, we found that the novel genistein derivative, [7-O-(2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl)-(1 → 4)-(6-O-acetyl-hex-2-ene-α-D-erythro pyranosyl)genistein, named G21, induced aberrations in mitotic spindle formation. In the presented study, we investigated the properties of G21 relevant to its genotoxic activity. The inhibition of topoisomerase IIα activity was evaluated in decatenation assay and immunoband depletion assay, the covalent DNA-topoisomerase IIα complexes and histone É£H2AX were detected immunofluorescently. Genotoxic effects of the tested compounds were assessed in micronucleation assay. The presence of centromeres in the micronuclei and the multiplication of centrosomes were evaluated in fluorescence immunolabelled specimens. The inhibition of tubulin polymerization was measured spectrophotometrically. We found that both tested drugs were able to inhibit topoisomerase II activity; however, G21, in contrast to genistein, blocked this enzyme at the concentration far exceeding cytotoxic IC(50). We also found that both compounds caused micronucleation in DU 145 prostate cancer cells, but in contrast to genistein, G21 exhibited aneugenic activity, manifested by the presence of centromeres in micronuclei formed in cells treated with the drug. Aneugenic properties of G21 resulted from the inhibition of tubulin polymerization and centrosome disruption, not observed in the presence of genistein. The study supports and extends our previous observations that the mechanisms of cytotoxicity of genistein and its new glycosidic derivative-G21 are significantly different.

Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells.

BACKGROUND: During functional studies on the rat stress-inducible Hspa1b (hsp70.1) gene we noticed that some liposome-based DNA carriers, which are used for transfection, induce its promoter activity. This observation concerned commercial liposome formulations (LA), Lipofectin and Lipofectamine 2000. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and practical consequences. RESULTS: We found that a reporter gene driven by Hspa1b promoter is activated both in the case of transient transfections and in the stably transfected cells treated with LA. Using several deletion clones containing different fragments of Hspa1b promoter, we found that the regulatory elements responsible for most efficient LA-driven inducibility were located between nucleotides -269 and +85, relative to the transcription start site. Further studies showed that the induction mechanism was independent of the classical HSE-HSF interaction that is responsible for gene activation during heat stress. Using DNA microarrays we also detected significant activation of the endogenous Hspa1b gene in cells treated with Lipofectamine 2000. Several other stress genes were also induced, along with numerous genes involved in cellular metabolism, cell cycle control and pro-apoptotic pathways. CONCLUSIONS: Our observations suggest that i) some cationic liposomes may not be suitable for functional studies on hsp promoters, ii) lipofection may cause unintended changes in global gene expression in the transfected cells.

Differential expression of HSPA1 and HSPA2 proteins in human tissues; tissue microarray-based immunohistochemical study.

In the present study we determined the expression pattern of HSPA1 and HSPA2 proteins in various normal human tissues by tissue-microarray based immunohistochemical analysis. Both proteins belong to the HSPA (HSP70) family of heat shock proteins. The HSPA2 is encoded by the gene originally defined as testis-specific, while HSPA1 is encoded by the stress-inducible genes (HSPA1A and HSPA1B). Our study revealed that both proteins are expressed only in some tissues from the 24 ones examined. HSPA2 was detected in adrenal gland, bronchus, cerebellum, cerebrum, colon, esophagus, kidney, skin, small intestine, stomach and testis, but not in adipose tissue, bladder, breast, cardiac muscle, diaphragm, liver, lung, lymph node, pancreas, prostate, skeletal muscle, spleen, thyroid. Expression of HSPA1 was detected in adrenal gland, bladder, breast, bronchus, cardiac muscle, esophagus, kidney, prostate, skin, but not in other tissues examined. Moreover, HSPA2 and HSPA1 proteins were found to be expressed in a cell-type-specific manner. The most pronounced cell-type expression pattern was found for HSPA2 protein. In the case of stratified squamous epithelia of the skin and esophagus, as well as in ciliated pseudostratified columnar epithelium lining respiratory tract, the HSPA2 positive cells were located in the basal layer. In the colon, small intestine and bronchus epithelia HSPA2 was detected in goblet cells. In adrenal gland cortex HSPA2 expression was limited to cells of zona reticularis. The presented results clearly show that certain human tissues constitutively express varying levels of HSPA1 and HSPA2 proteins in a highly differentiated way. Thus, our study can help designing experimental models suitable for cell- and tissue-type-specific functional differences between HSPA2 and HSPA1 proteins in human tissues.

Synthetic conjugates of genistein affecting proliferation and mitosis of cancer cells.

This paper describes the synthesis and antiproliferative activity of conjugates of genistein (1) and unsaturated pyranosides. Constructs linking genistein with a sugar moiety through an alkyl chain were obtained in a two-step synthesis: in a first step genistein was converted into an intermediate bearing an ω-hydroxyalkyl substituent, containing two, three or five carbon atoms, at position 7, while the second step involved Ferrier glycosylation reaction, employing glycals. Antiproliferative activity of several genistein derivatives was tested in cancer cell lines in vitro. The most potent derivative, Ram-3 inhibited the cell cycle, interacted with mitotic spindles and caused apoptotic cell death. Neither genistein nor the sugar alone were able to influence the mitotic spindle organization. Our results indicate, that conjugation of genistein with certain sugars may render the interaction of derivatives with new molecular targets.

HSPA2 Chaperone Contributes to the Maintenance of Epithelial Phenotype of Human Bronchial Epithelial Cells but Has Non-Essential Role in Supporting Malignant Features of Non-Small Cell Lung Carcinoma, MCF7, and HeLa Cancer Cells.

Heat Shock Protein A2 (HSPA2) is a member of the HSPA (HSP70) chaperone family and has a critical role for male fertility. HSPA2 is present in a number of somatic organs. Limited evidence suggests that HSPA2 may be involved in regulating epithelial cell differentiation. HSPA2 also emerged as a cancer-related chaperone; however, no consensus on its functional significance has been reached so far. In this study, we compared the phenotypic effects of HSPA2 deficit in non-transformed human bronchial epithelial cells (HBEC), and in lung, breast, and cervical cancer cells. We used various techniques to inhibit the HSPA2 gene expression in order to examine the impact of HSPA2 deficiency on cell growth, migration, adhesion, and invasion. Our results show that HBEC but not cancer cells are sensitive to HSPA2 deficit. HSPA2 knockdown in HBEC cells impaired their clone-forming ability and adhesiveness. Thus, our results indicate that epithelial cells can rely on a specific activity of HSPA2, but such dependence can be lost in epithelial cells that have undergone malignant transformation.

Unsaturated genistein disaccharide glycoside as a novel agent affecting microtubules.

Genistein, due to its recognized chemopreventive and antitumor potential, is a molecule of interest as a lead compound in drug design. While multiple molecular targets for genistein have been identified, so far neither for this isoflavonoid nor for its natural or synthetic derivatives disruption of microtubules and mitotic spindles has been reported. Here we describe such properties of the synthetic glycosidic derivative of genistein significantly more cytotoxic than genistein, 7-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->4)-(6-O-acetyl-hex-2-ene-alpha-D-erythro-pyranosyl)genistein, shortly named G21. We found that G21 causes significant mitotic delay, frequent appearance of multipolar spindles, and alteration of the interphase microtubule array.

Heat shock proteins in the physiology and pathophysiology of epidermal keratinocytes.

Heat shock proteins (HSPs), a large group of highly evolutionary conserved proteins, are considered to be main elements of the cellular proteoprotection system. HSPs are encoded by genes activated during the exposure of cells to proteotoxic factors, as well as by genes that are expressed constitutively under physiological conditions. HSPs, having properties of molecular chaperones, are involved in controlling/modulation of multiple cellular and physiological processes. In the presented review, we summarize the current knowledge on HSPs in the biology of epidermis, the outer skin layer composed of stratified squamous epithelium. This tissue has a vital barrier function preventing from dehydratation due to passive diffusion of water out of the skin, and protecting from infection and other environmental insults. We focused on HSPB1 (HSP27), HSPA1 (HSP70), HSPA2, and HSPC (HSP90), because only these HSPs have been studied in the context of physiology and pathophysiology of the epidermis. The analysis of literature data shows that HSPB1 plays a role in the regulation of final steps of keratinization; HSPA1 is involved in the cytoprotection, HSPA2 contributes to the early steps of keratinocyte differentiation, while HSPC is essential in the re-epithelialization process. Since HSPs have diverse functions in various types of somatic tissues, in spite of multiple investigations, open questions still remain about detailed roles of a particular HSP isoform in the biology of epidermal keratinocytes.

Functional redundancy of HSPA1, HSPA2 and other HSPA proteins in non-small cell lung carcinoma (NSCLC); an implication for NSCLC treatment.

Heat shock proteins (HSPs) are a large group of chaperones considered critical for maintaining cellular proteostasis. Their aberrant expression in tumors can modulate the course of processes defined as hallmarks of cancer. Previously, we showed that both stress-inducible HSPA1 and testis-enriched HSPA2, highly homologous members of the HSPA (HSP70) family, are often overexpressed in non-small cell lung carcinoma (NSCLC). HSPA1 is among the best characterized cancer-related chaperones, while the significance of HSPA2 for cancer remains poorly understood. Previously we found that in primary NSCLC, HSPA1 was associated with good prognosis while HSPA2 correlated with bad prognosis, suggesting possible different roles of these proteins in cancer. Therefore, in this work we investigated the impact of HSPA1 and HSPA2 on NSCLC cell phenotype. We found that neither paralog-selective nor simultaneous knockdown of HSPA1 and HSPA2 gene expression reduced growth and chemoresistance of NSCLC cells. Only blocking of HSPA proteins using pan-HSPA inhibitors, VER-155008 or JG-98, exerted potent anticancer effect on NSCLC cells, albeit the final outcome was cell type-dependent. Pan-HSPA inhibition sensitized NSCLC cells to bortezomib, but not to platinum derivates. Our result suggests the inhibitors of proteasome and HSPAs seem an effective drug combination for pre-clinical development in highly aggressive NSCLC.

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

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.

Stress proteins HSP27 and HSP70i predict survival in non-small cell lung carcinoma.

BACKGROUND: The expression pattern of stress (heat shock) proteins (HSPs) in cancer cells is frequently different from that observed in normal cells; most often some stress-inducible HSPs are constitutively and highly expressed. The objective of this study was to determine the prognostic significance of stress proteins HSP70i and HSP27 in non-small cell lung carcinoma (NSCLC). MATERIALS AND METHODS: An immunohistochemical procedure that enables unambiguous detection of HSP70i protein was used. RESULTS: Strong HSP70i staining showed a survival advantage, although multivariate analysis did not confirm this result. There was an evident correlation between HSP27 overexpression and survival of patients and the results were confirmed by multivariate analysis: 70% of patients with HSP27-negative tumors died within one year after the surgery. CONCLUSION: Our data suggest that HSP27 and HSP70i positivity may represent a favorable prognostic factor in NSCLC.

Oligo(3-hydroxybutanoate) conjugates with acetylsalicylic acid and their antitumour activity.

In this paper we discuss the anticancer activity of acetylsalicylic acid with oligo(3-hydroxybutanoate) conjugates, their characteristics and in vitro biological evaluation. Acetylsalicylic acid (aspirin) attached via hydrolysable ester bonds to non-toxic well-defined 3-hydroxybutanoic acid oligomers shows novel method of drug modification. The resulting conjugates were more effective than aspirin in growth inhibition of human colon adenocarcinoma cells HT-29 and human colon carcinoma cells HCT 116 in vitro. Treatment of rats with doses as high as 2g of the conjugate (equivalent to 0.6g of pure aspirin) per kg of body weight did not exhibit toxic effects.

The Role of Heat Shock Proteins in Cisplatin Resistance.

BACKGROUND: Cisplatin (CDDP), a small molecule platinum-based compound, is an effective anticancer drug used against a wide range of human neoplasms. Long-term clinical use of CDDP is however limited due to the development of drug resistance and the possible incidence of serious side effects including nephrotoxicity and ototoxicity. The mechanisms underlying resistance of cells to CDDP are complex, and among them, the cytoprotective involvement of proteins referred to as Heat Shock Proteins (HSP) seems potentially important. METHODS: We searched various electronic databases including PubMed and selected the reports concerning the contribution of HSPs to CDDP resistance of cancer cells and to minimize the CDDP-induced nephrotoxicity and ototoxicity. RESULTS: This critical review of data collected so far summarizes the results on the major HSPs: HSP27/HSPB1, HSP70/HSPA1, HSP90/HSPC and GRP78/HSPA5, because only these have been the subject of the most intense research in the matter discussed here. We also provide relevant information concerning some other HSPs, namely HSPA9/mortalin, HSPA2, HSP110 and DNAJ. A possible role of HSPs in counteracting CDDP-induced neprho- and ototoxicity is mentioned. CONCLUSIONS: This review shows that no universal relationship between the levels of expression of HSPs and sensitivity of cancer cells to CDDP can be confirmed. Multiple observations indicate however that such correlation can rather manifest as a molecular or cellular context-dependent phenomenon. Thus, HSPs can be viewed as an important component of the multifactorial, complex response of cancer cells to CDDP. However, to strengthen such a conviction, more extensive studies are needed.

The structural and functional analysis of the human HSPA2 gene promoter region.

HSPA2 is a human counterpart of the testis-specific rodent Hst70/Hsp70.2 gene. In contrast to the latter, the expression of the human HSPA2 gene is not limited to the testis, and recent data show that human tumor cells can express this gene at significant levels. The characteristics of HSPA2 expression suggests that it can influence the phenotype and survival of cancer cells similarly as overexpression of major members of the HSP70 gene family. Until now, neither the structure of the transcription unit of the human HSPA2 gene has been established nor a functional analysis of its promoter performed. In this study we established that the human HSPA2 gene, in contrast to its rodent counterparts, is intronless and has a single transcription start site. We also show that the same type of HSPA2 transcripts are synthesized in the testes and in cancer cell lines. In order to perform a functional study of the HSPA2 promoter, we used a transient transfection assay and found that the 392 bp fragment upstream of the ATG codon was a minimal region required for efficient transcription, while a 150 bp deletion from the 5' end of this region dramatically reduced the promoter activity. Delineation of the minimal promoter is a basic step toward identifying the cis and trans elements involved in the regulation of the HSPA2 gene expression in cancer cells.

The GC-box is critical for high level expression of the testis-specific Hsp70.2/Hst70 gene.

The Hsp70.2/Hst70 gene, which belongs to the 70 kDa heat-shock protein (HSP) family, is expressed specifically in primary spermatocytes and spermatids. The regulatory elements required for a high level of testis-specific expression of the gene are placed between the two major transcription start sites T1 and T2 (approximately 350 and 115 bp upstream of the starting ATG codon). Here we have shown that sequences proximal to the exon1/intron splicing site in the 5' untranslated region of the Hsp70.2/Hst70 gene, which include a highly conserved element called box B, are required for efficient expression of the chloramphenicol acetyltransferase reporter gene in testes of transgenic mice. However, in spite of the drastically reduced overall activity, the stage-specific expression pattern of the transgene was preserved after removal of these sequences. We have also shown that GC-box located downstream of the box B (approximately 210 bp upstream of the starting ATG codon) is indispensable for efficient expression of the Hsp70.2/Hst70 gene promoter in spermatogenic cells. The GC-box specifically binds proteins present in nuclear extracts from testes (putatively Sp1-like factors). A change in the pattern of such GC-box-interacting factors corresponds to activation of the Hsp70.2/Hst70 gene, confirming the importance of this regulatory element.

Immunohistochemical detection of the inducible heat shock protein hsp70: a methodological study.

Stress-inducible Hsp70i and constitutively expressed Hsc70 are highly related heat shock proteins. Aberrant expression levels and intracellular localization of these proteins has been suggested as a potential marker in certain tumors. The aim of our study was to work out a reliable, immunohistochemical detection of the stress-inducible Hsp70i protein and enabling discrimination between Hsp70i and Hsc70 proteins in paraffin-embedded human tissues. We tested the effect of several fixative procedures and antigen retrieval on the effectiveness of the Hsp70i detection in murine cells cultured in vitro and in liver of rats subjected to heat shock. For cells grown in vitro, specific Hsp70i immunoreactivity was obtained with all fixatives used. However, samples fixed in 10% formalin and 4% paraformaldehyde required antigen retrieval. In liver tissue embedded in paraffin, regardless the fixative used, positive Hsp70i staining could be visible only if antigen retrieval was applied. We applied this procedure for detection of Hsp70i in routine sections of breast and lung cancers fixed with 10% formalin and found that the application of thermal antigen retrieval significantly enhanced the SPA810 immunoreactivity and reduced background staining. This procedure enabled also the differential detection of Hsp70i and Hsc70 in routine histopathological preparations.