Monoclonal antibodies to mammalian heat shock proteins impair mouse embryo development in vitro

Two-cell mouse embryos (B6D2F1) were cultured in the presence or absence of 100 microg/ml monoclonal antibodies specific for the mammalian 60 kDa (HSP60), 70 kDa (HSP70) and 90 kDa (HSP90) heat shock proteins. Embryo development was evaluated after 3, 5 and 7 days in culture by determining the number of blastocysts, hatched blastocysts and outgrown trophoblasts at the successive time points. At day 3, only 29% (22/75) of the embryos cultured with anti-HSP60 antibody developed to the blastocyst stage (P < 0.0001) as compared to 67% (31/46) of the embryos cultured with anti-HSP70, 72% (43/60) cultured with anti-HSP90, and 79% (49/62) in medium plus mouse IgG1. By day 5, hatched embryos were present in 28% (13/ 46) of the cultures containing anti-HSP70 (P < 0.0001), as opposed to 57% (34/60) containing anti-HSP90 and 73% (45/62) containing IgG1. At day 7, outgrown trophoblasts were observed in 9% (4/46) of cultures containing anti-HSP70 (P < 0.0001), 45% (27/60) containing anti-HSP90 (P < 0.01) and 66% (41/62) cultured in medium plus IgG1. Antibodies to different heat shock proteins exerted a detrimental effect on mouse embryo development at unique development stages. Immune sensitization to heat shock proteins may be a cause of reproductive failure.     

Regulation of megakaryocytopoiesis and platelet production: lessons from animal models

We showed differential expression of HSP70 during oral tumorigenesis. The precise functional role of HSP70 overexpression in the pathogenesis of betel and tobacco related oral cancer remains to be determined. To evaluate the utility of HSP70 as an indicator of the biological stress experienced by tumour cells or the malignant potential of oral epithelial lesions and predicting clinical outcome, its expression was assessed in different stages of oral carcinogenesis by immunohistochemical analysis and correlated with clinicopathological parameters. Overexpression of HSP70 protein was observed in 38 of 64 (59%) dysplastic lesions and 92 of 125 (74%) oral squamous cell carcinomas (SCCs) which included 76 of 105 cases (72%) of primary oral SCCs and 16 of 20 (80%) of recurrent oral SCCs. A significant correlation of HSP70 expression was observed with severity of dysplasia (P = 0.0006767), poor histological differentiation of primary tumours (P = 0.0184348), increase primary tumour size (P = 0.0221103) and consumption of betel and tobacco (P < 0.01). Follow-up studies showed that in patients with premalignant lesions the median transition time (premalignancy to malignancy) was significantly shorter in HSP70 overexpressing cases than those showing basal level of HSP70 (P = 0.012). Oral cancer patients with elevated levels of HSP70 showed decreased median disease-free survival time (no recurrence/metastasis) than those showing basal HSP70 immunoreactivity (P = 0.0246). The results suggest that HSP70 expression may not be a mere marker of biological stress but may also be implicated in the pathogenesis of oral cancer.     

Inhibition of ischaemic tolerance in the gerbil hippocampus by quercetin and anti-heat shock protein-70 antibody

To clarify the role of heat shock protein-70 (HSP70) in ischaemic tolerance following pretreatment with sublethal cerebral ischaemia, we examined whether the induction of tolerance in the gerbil hippocampus is inhibited by quercetin, an inhibitor of HSP70 expression, or anti-HSP70 antibody. A 3 min period of forebrain ischaemia was induced following pretreatment with 2 min of ischaemia and 3 days of reperfusion. Quercetin or anti-HSP70 antibody was continuously infused into the left lateral ventricle using an implanted osmotic minipump started 3 h after or 2 h before the first ischaemia. The animals were killed 4 days after the second ischaemia for histological observations. Both agents produced no neuronal damage in the brain following a single 2 min period of ischaemia. The neuronal density of the CA1 hippocampus in animals subjected to treatment with quercetin and anti-HSP70 antibody was significantly lower than vehicle-treated animals but were significantly higher than animals with a single 3 min period of ischaemia. Thus, the present study showed that quercetin and anti-HSP70 antibody prevent the induction of ischaemic tolerance. The result suggests that HSP70 expression, at least in part, plays a role in the induction of ischaemic tolerance.     

Heart rate variability in insomniacs and matched normal sleepers

BACKGROUND: Heat shock proteins have been associated with the mutant form of the tumor suppressor gene, TP53, and with resistance to cancer chemotherapy. METHODS: Archival tissues from 50 patients with head and neck squamous cell carcinoma who received primary surgical resection were examined for p53, HSP27, and HSP70 by immunohistochemistry and correlated with tumor stage, grade, and 5-year survival (alive or deceased). RESULTS: Both heat shock proteins were strongly expressed in normal mucosa and in small (T1 and T2) tumors. Thirty (60%) of tumors were positive for p53, 43 (86%) for HSP27, and 34 (68%) for HSP70, with no association between p53 and heat shock protein expression. Twenty-five patients were alive (4 with disease), and 25 patients were deceased (9 from other causes). p53 Protein overexpression correlated with low-grade tumors. Only primary tumor site (i.e., oral cavity > larynx > oropharynx/base of tongue) and N stage were significantly associated with survival. CONCLUSIONS: Heat shock proteins are expressed in normal upper respiratory tract squamous mucosa, and their role in carcinoma remains unclear. None of the markers, p53, HSP27, or HSP70, demonstrated prognostic significance for 5-year survival. We confirm the recognized association of cervical lymph node metastases with decreased survival.     

Jack fruit lectin-specific glycoconjugate expression during the progression of cervical intraepithelial neoplasia: a study on exfoliated cells

The expression of glycoconjugates specific to Jack fruit lectin (JFL) was studied in the exfoliated squamous cells of different grades of intraepithelial and invasive neoplasia of the uterine cervix. It was observed that while normal cells showed almost negative binding, the lectin binding percentage of squamous cells significantly increased with increasing atypia of the epithelium. Correlation analysis between different groups revealed that mild lectin binding in cells had a negative correlation and intense binding had a positive correlation with various stages of tumor progression. These results indicate that the number of cells with aberrant expression of glycoconjugates increases as neoplastic transformation advances. The percentage of labeled and unlabeled cells also shows a continuous transition from low to severe grades of cervical intraepithelial neoplasia and invasive carcinomas. The present study therefore shows that JFL may be used as a probe for further elaboration of detection and grading of precancerous and cancerous lesions of the uterine cervix.     

Heterogeneous patterns of constitutive and heat shock induced expression of HLA-linked HSP70-1 and HSP70-2 heat shock genes in human melanoma cell lines

The heat shock response, which is characterized by the induction of heat shock proteins, is known to affect the ability of tumour cells to cope with potentially adverse conditions such as hypoxia, glucose starvation and cytotoxic immune reactions. To assess the heat shock response of melanoma cells, spontaneous and heat shock induced expression of heat shock proteins was analysed in a panel of 17 human melanoma cell lines. Constitutive expression of HSP27, HSP70, HSC70, HSP90alphabeta and GRP94 proteins was found in all the melanoma cell lines, and HSP70 and HSC70 were also induced by heat shock. The major heat inducible HLA-linked HSP70-1 and HSP70-2 genes were analysed at the mRNA level. Basal expression and inducibility varied between the different melanoma cell lines. In addition, in situ hybridization demonstrated heterogeneous expression of these genes among single cells of a given cell line. In general, each melanoma cell line appears to exhibit an individual type of HSP70 expression that might reflect selection during tumour progression and therapy.     

Reduced induction of HSP70 in PC12 cells during neuronal differentiation

Rat pheochromocytoma PC12 cells differentiate into nonreplicating neuronal cells with neurite extensions in response to nerve growth factor (NGF). To gain better understanding of the regulation of stress responses in neuronal cells, we examined the induction of HSP70, HSP70 mRNA, and heat shock factor 1 (HSF1) DNA-binding activity following treatment by heat shock or with sodium arsenite or amino acid analogue in PC12 cells treated with or without NGF. The induction of HSP70 and HSP70 mRNA following these stresses was diminished in the differentiated PC12 cells compared to the undifferentiated cells, whereas the HSF1 DNA-binding activity was enhanced in the differentiated PC12 cells. This phenomenon was characteristic of the differentiated neuronal cells rather than growth-arrested cells. Thus, neuronal cells appear to show an altered stress response depending on their differentiation state, and the diminished HSP70 expression in the differentiated neuronal cells may explain the sensitivity of neuronal cells to pathophysiological stressors.     

Multiple sclerosis: altered expression of 70- and 27-kDa heat shock proteins in lesions and myelin

Recent studies have implicated heat shock proteins (HSP) in the pathogenesis of the multiple sclerosis (MS) lesion. Expression of the 73 kDa constitutive HSP (HSC70), the 72 kDa stress-inducible HSP (HSP70), and the 27 kDa small HSP (HSP27) was analyzed in white matter and myelin from central nervous system (CNS) tissue of MS and normal subjects using a combination of immunocytochemistry and quantitative immunoblotting. Plaques of all types were sharply defined by reduced immunostaining for HSC70, and shown by immunoblotting to contain 30 to 50% less HSC70 than surrounding white matter or normal tissue. In contrast, HSP27 was markedly enhanced 2.5- to 4-fold in plaque regions, especially in fibrous astrocytes and in hyperplastic interfascicular oligodendrocytes at the lesion edge. HSP70 was less abundant than HSC70, and no significant differences in HSP70 levels were noted between MS and normal white matter. Myelin isolated from active plaques contained 3- to 4-fold more HSC70 than normal myelin. Pronounced expression of HSP70 and HSP27 was also found in MS myelin, although neither protein was detected in normal myelin. Thus, white matter undergoing immune-mediated destruction in MS was associated with altered distribution and expression of HSC70 and HSP27. These changes may initially serve to protect myelin from further destruction and facilitate repair; however, enhanced expression of HSC70, HSP70, and HSP27 in myelin may subsequently present as additional immune targets involved in the progression of disease.     

Histopathologic spectrum of vaginal adenosis and related changes in stilbestrol-exposed females

A total of 98 colposcopically directed biopsies were obtained from the vagina, cervix, and cervicovaginal ridge (hood) of 80 young women believed to have had intrauterine exposure to stilbestrol (DES). Specific investigation of the patient's medical records corroborated the history of maternal stilbestrol administration in 36 patients (45%), while in the remainder the drug history was regarded as presumptive since medical records were unavailable for review. The findings did not differ significantly in those biopsies taken from patients with confirmed or presumptive drug histories. Histologic evidence of vaginal adenosis was detected in vaginal biopsies from 43 patients. In 30 cases (70%) benign Müllerian-type glandular epithelium was in the superficial vaginal wall, residing on the mucosal surface and/or in the lamina propria. The glandular epithelium predominantly was of endocervical type, but in six instances it resembled endometrial or fallopian tubal epithelium. The glands were accompanied by varying degrees of squamous metaplasia in 22 cases. When extensive the metaplasia produced transformation zones similar to those seen in the normal cervix. Vaginal biopsies of adenosis from the other 13 patients (30%) revealed squamous metaplasia without demonstrable glands due to complete transformation of all antecedent glandular epithelium by squamous metaplasia. Our studies indicate that squamous metaplasia is a component of major importance in the natural history of adenosis and that the concept of adenosis should be broadened to include those examples comprised exclusively of metaplastic epithelium. In such examples metaplasia is identified by the immaturity and poor glycogenation of the squamous cells and their accompanying squamous pegs which often contain residual gland openings or squamous "eddies." Similar findings were present in biopsies of seven cervicovaginal ridges and in cervical biopsies from 37 patients, except for the absence of endometrial or tubal type glands in the latter site. Although no adenocarcinomas were detected, six patients had squamous dysplasia of the vagina and/or cervix. In no case were premalignant or dysplastic changes of glandular cells found. Our findings support the thesis that stilbestrol-associated adenosis represents anomalous embryologic localization of the original squamocolumnar junction in the vagina rather than in the cervix. It is closely related to so-called cervical "erosions." The development of squamous metaplasia accounts for modifications in the clinical and histologic appearances by producing transformation zones which then may be subject to the same oncogenic stimuli for squamous neoplasia as are their counterparts in the cervix.     

Small-cell neuroendocrine carcinoma of the uterine cervix associated with micro-invasive squamous cell carcinoma and adenocarcinoma in situ

A case of small-cell neuroendocrine carcinoma of the uterine cervix associated with squamous cell carcinoma and adenocarcinoma in situ is reported. The tumor consisted mainly of uniform small cells with a population of intermediate cells that resembled carcinoid tumor cells. Foci of micro-invasive squamous cell carcinoma and adenocarcinoma in situ were recognized separately, adjacent to the main tumor. Both Grimelius stain and immunostaining of serotonin were positive for small-cell and intermediate-cell carcinoma. Neurosecretory granules were demonstrated by electron microscopy. Microacini with positive mucin staining and microvilli-like structures suggested glandular or exocrine differentiation of the tumor. Three distinctive types of differentiation, neuroendocrine, exocrine and squamous characteristics, were expressed in the tumor.     

Increased expression of cyclooxygenase 2 occurs frequently in human lung cancers, specifically in adenocarcinomas

Cyclooxygenase (COX)-2 expression was immunohistochemically examined in 59 human lung cancers as well as in normal and premalignant lung specimens. In contrast to scattered weak reactivity seen in normal peripheral airway epithelial cells, markedly up-regulated COX-2 expression was detected in about one-third of atypical adenomatous hyperplasias and carcinoma in situ specimens, and a significant increase in COX-2 expression was observed in 70% of invasive adenocarcinoma cases. Interestingly, the proportion of adenocarcinoma cells with marked COX-2 expression was much greater in lymph node metastases than in the corresponding primary tumors. In contrast, small cell carcinomas showed virtually negligible expression, and squamous cell carcinomas showed infrequent and low expression. These findings suggest that an increase in COX-2 expression may be associated with the development of adenocarcinomas and possibly with acquisition of an invasive and metastatic phenotype.     

Absence of epidermal growth factor receptor expression in squamous cell carcinoma of the uterine cervix is an indicator of limited tumor disease

The expression of growth factors is considered as an important diagnostic and prognostic feature in tumor pathology. We investigated the value of the immunohistochemical EGF-receptor expression (EGF-R) in 30 squamous cell carcinomas of the uterine cervix, treated by radical hysterectomy and lymphadenectomy according to the Wertheim-Meigs-Okabayashi technique. Immunohistochemical reactions were performed on 4 microm sections from paraffin-embedded tissue, using an indirect peroxidase method. The staining results were evaluated semiquantitatively as negative (n=9; 30%) or as slightly, moderately or severely positive (n=21; 70%). The EGF-R-negative tumors were found in less advanced tumor stages. None had invaded into the parametrium (100%), eight were staged as T1 (89%), seven as N0 (78%), and seven showed no evidence for lymphangiosis carcinomatosa (78%). The respective values for the EGF-R-positive tumors ranged from 52% to 67%. However, only the difference in parametral invasion (EGF-R-negative: 0%, EGF-R-positive: 38%) was statistically significant (p=0.0306), probably due to the small number of cases. The EGF-R-expression was not correlated to histomorphological tumor grading. The results of this study indicate an inverse correlation between EGF-R expression and tumor spread. Assuming that this trend could be confirmed by a larger group of patients, immunostaining for EGF-R in a tumor biopsy could be useful to adapt surgical strategies and adjuvant therapy in the individual patient. Moreover, the EGF-R is an interesting target for immunotherapeutic approaches in squamous cell cervical carcinoma.     

Evidence that GABA transmission mediates context-specific extinction of learned fear

Recent evidence has suggested that molecular chaperones participate in the conformational change between the normal cellular prion protein (PrPC) and its scrapie isoform (PrPSc). To study a role of PrPC in the regulation of expression of heat shock proteins (HSPs), a group of molecular chaperones, heat-induced expression of major HSPs (HSP105, HSP90alpha, HSP72, HSC70, HSP60, and HSP25) was investigated in cultured skin fibroblasts isolated from the mice homogeneous for a disrupted PrP gene (PrP-/- mice) by Western blot analysis and immunocytochemistry. Two lines of fibroblasts were established and designated SFK derived from the PrP-/- mice and SFH derived from the PrP+/+ mice, respectively. In both SFK and SFH cells, HSP105, HSP72, and HSP25 were expressed at low levels under unstressed conditions but they were induced markedly following exposure to heat stress (43 degreesC/20 min) at 3-72 h postrecovery. In both cell types, HSC70 and HSP60 were expressed at high levels under unstressed conditions and their levels remained unchanged after heat shock treatment. HSP90alpha was undetectable in both cell types under any conditions examined. The pattern of expression, induction, and subcellular location of HSP105, HSP72, HSC70, HSP60, and HSP25 was not significantly different between SFK and SFH cells under unstressed and heat-stressed conditions. Furthermore, the levels of constitutive expression of HSP105, HSC70, HSP60, and HSP25 were similar between the brain tissues isolated from the PrP-/- and PrP+/+ mice. These results indicate that HSP induction is not affected by either the existence or the absence of PrPC in the cells.     

Expression of Tn and sialyl-Tn antigens in the neoplastic transformation of uterine cervical epithelial cells

The expression of simple mucin-type carbohydrate antigens, Tn and sialyl-Tn antigens, was evaluated by immunohistochemical staining with monoclonal antibodies in normal squamous epithelium, dysplasia, carcinoma in situ, and invasive squamous cell carcinoma of the uterine cervix. The expression of the Tn antigen detected by HB-Tn1 and B1.1 was found in 17 (20%) and 19 (23%) of the 83 invasive carcinomas, respectively, but was not found in the 36 normal squamous epithelia, 22 severe dysplasias, or 24 carcinomas in situ. The sialyl-Tn antigen was detected by HB-STn1 and TKH-2 in 14 (64%) and 11 (50%) of the 22 severe dysplasias, 13 (54%) and 10 (42%) of the 24 carcinomas in situ and 48 (58%) and 42 (51%) of the 83 invasive carcinomas, respectively, but was completely absent in 36 normal squamous epithelia. Coexpression of the sialyl-Tn antigen was observed in 89% of the cases expressing the Tn antigen. No significant difference was observed between the immunoreactivities of the antigens in the metastatic lymph nodes and primary tumors. No correlation was found between the expression of each antigen and clinical state, histologic type, depth of invasion, parametrial spread, lymphatic and vessel permeation, lymph node metastasis, or 5-year survival rate. The expression of Tn and sialyl-Tn demonstrates a specific change in the neoplastic progression from carcinoma in situ to invasive carcinoma and from normal to dysplasia, respectively, in squamous cell neoplastic lesions of the cervix. Tn and sialyl-Tn antigens may be useful markers for biologic investigation of neoplastic transformation in cervical squamous cell carcinoma.     

Heat shock protein 27 enhances the tumorigenicity of immunogenic rat colon carcinoma cell clones

The REG and PRO cell clones were obtained from a colon adenocarcinoma induced in a BDIX rat by 1,2-dimethylhydrazine. When injected s.c. into syngeneic hosts, REG cells induce tumors that regress in less than 3 weeks, whereas PRO cells, like parental cells, induce progressive tumors. Here, we show that compared to PRO cells, REG cells are more sensitive to cell death induced by anticancer drugs. The small heat shock protein (HSP) 27 is not expressed or inducible in REG clones, whereas it is abundantly expressed and inducible by heat shock in PRO clones. The expression of HSP27 in REG cells increases their resistance to apoptosis in vitro and dramatically enhances their tumorigenicity when injected s.c. into syngeneic rats. HSP27 expression in REG cells both increases tumor size and delays tumor regression. This increased tumorigenicity is associated with a substantial decrease of in vivo tumor cell apoptosis. We conclude that HSP27 expression in malignant cells increases their tumorigenicity in syngeneic animals. In combination with the role of HSP27 in tumor cell resistance to cytotoxic agents, its contribution to tumorigenicity makes this protein a potential target for antitumoral therapy.     

The influence of carbon source on the level and composition of ceramides of the Candida lipolytica yeast

AIMS AND BACKGROUND: Altered oncogenic activity is a feature associated with many malignant and premalignant conditions. Among the many oncogenes, ras and myc are commonly altered in many tumors. This study aims to evaluate the expression of ras and c-myc oncoproteins in a total of 204 cervical tissue samples, including premalignant and malignant lesions as well as apparently normal cervical tissue. METHODS AND STUDY DESIGN: Mouse monoclonal antibodies against the three mammalian ras gene products (c-H-ras, c-K-ras, c-N-ras) and the c-myc protein were used to evaluate oncoprotein expression by immunocytochemistry. RESULTS: None of the samples analyzed displayed immunoreactivity for H-ras and K-ras. Normal cervical epithelium showed minimal immunoreactivity for N-ras with about 33% of the samples expressing the protein. More conspicuous expression in normal tissue was displayed by c-myc, with about 90% of the samples expressing the protein (mean value of cells positive=34%). The immunoreactivity for N-ras increased with increasing histological abnormality from low-grade squamous intraepithelial lesions (SIL) to invasive carcinoma. Increased immunoreactivity for N-ras was evident in the basaloid cells of malignant lesions, with the maximum value of 66% found in poorly differentiated squamous cell carcinoma (PDSCC). The percentage of nuclei positive for c-myc also showed a gradual increase from low-grade SIL onwards, the highest positivity being found in PDSCC, where the mean value was 85%. Statistical analysis revealed a good correlation between the expression of N-ras (r=0.8922, P=0.001) and c-myc (r=0.8856, P=0.001) and various histological stages of tumor progression in the cervical epithelium. CONCLUSIONS: These results therefore suggest that c-myc and N-ras oncoproteins are important during tumor progression in the uterine cervix.     

Intestinal expression of human heat shock protein 90 in patients with Crohn's disease and ulcerative colitis

Heat shock proteins are induced by several stress factors and are potential antigens in autoimmune disorders. Expression of heat shock protein 90 (HSP 90) was investigated in patients with inflammatory bowel disease and normal controls. We combined western blot analysis with laser densitometry for quantitation. Localization of HSP 90 was investigated by immunohistochemistry. Western blots showed a significant mucosal expression of HSP 90, which was comparable in patients and controls. There was also no difference between normal and inflamed mucosa in inflammatory bowel disease. In immunohistochemical staining studies, HSP 90 was detected in epithelial cells, mononuclear cells, giant cells, nerve cells, and endothelial cells of small vessels. There was no difference in the intensity of staining or localization in patients with inflammatory bowel disease compared to controls. These findings render a potential protective or immunogenic function of HSP 90 in inflammatory bowel disease unlikely.     

Concomitant alterations in distribution of 70 kDa heat shock proteins, cytoskeleton and organelles in heat shocked 9L cells

Maintenance of cell architecture and positioning of organelles are major functions of the cytoskeleton. On the other hand, induction of heat shock proteins (HSPs) and reorganization of the cytoskeleton are the most significant changes in heat-shocked mammalian cells. We examine the alterations in HSP70 and its constitutively expressed cognate, HSC70, as well as the cytoskeleton and organelles in 9L rat brain tumor cells upon heat shock. We employed fluorescence microscopy and scanning electron microscopy to follow these changes. Levels of HSP70s were quantified by Western blotting. Accumulation of HSC70 was more transient and the protein translocated to and subsequently exited from the nucleus more rapidly than HSP70. Changes in actin microfilaments include the nuclear localization of actin fraction and disappearance of cytoplasmic microfilament bundles, while the cortical actin microfilaments were almost unaffected. Furthermore, microtubules retracted slightly from the cell periphery but remained largely unchanged. In contrast, the intermediate filaments collapsed into the perinuclear region. The mitochondria converted from filamentous into granular forms and clustered in a region overlapping with the collapsed intermediate filaments. All of the above alterations are reversible and largely reverted after 8 h of recovery. The effect on Golgi organization was very transient and the apparatus assumed a normal appearance within 4 h after the heat treatment. The ER, on the other hand, was totally unaffected by the heat treatment. These observations help correlate the sequential events following a stress like heat shock and suggest possible physiological functions of these essential constituents of a cell under stress.     

The use of thrombomodulin to study epithelial cell differentiation in neoplastic and non-neoplastic oral lesions

Thrombomodulin (TM) is a glycoprotein originally isolated from rabbit lung vasculature and characterized as a natural endothelial anticoagulant. Thrombin binds to TM noncovalently with high affinity. Thrombin-TM complexes can activate protein C efficiently. Activated protein C inactivates factors Va and VIIIa and regulates the blood coagulation cascade. Thus TM converts thrombin from a procoagulant protease to an anticoagulant. TM is found on endothelial cells in veins, arteries and capillaries. Our previous study has shown that TM is also expressed on the cell surface of squamous epithelium. In the present study, we aimed to disclose differences in TM expression among normal, dysplastic, and malignant squamous epithelium in human oral mucosa by counting TM-positive cells in each lesion. TM was uniformly expressed in the spinous layer of normal human oral squamous epithelium. The number of TM-positive cells was not significantly different between normal epithelium, lichen planus and mild dysplasia. In contrast, in moderate and severe dysplasia and well-differentiated squamous cell carcinoma (SCC), there were significantly fewer positive cells compared with normal epithelium. In SCCs, the periphery and the central keratinized area of tumor islands were often negative. The proportion of TM-positive cells in poorly differentiated SCC was significantly lower than in well-differentiated SCC. These results indicate that TM may have diagnostic value in the histological examination of oral premalignant and malignant lesions.     

Heat shock protein 72 modulates pathways of stress-induced apoptosis

The resistance to stress-induced apoptosis conferred by the thermotolerant state or by exogenous expression of HSP72 was measured in mouse embryo fibroblasts. The induction of thermotolerance protects cells from heat, tumor necrosis factor alpha (TNFalpha), and ceramide-induced apoptosis but not from ionizing radiation. Because the development of thermotolerance is associated with increased levels of heat shock proteins, we determined whether constitutive expression of one of the major inducible heat shock proteins, HSP72, could also protect cells from stress-induced apoptosis. Cells expressing constitutive HSP72 were shown to have significantly reduced levels of apoptosis after heat, TNFalpha, and ceramide but not after ionizing radiation. Activation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) was found to be strongly inhibited in thermotolerant cells after heat shock but not after other stresses. Cells that constitutively express HSP72 did not demonstrate decreased SAPK/JNK activation after any of these stresses. Thus, factors other than HSP72 that are induced in the thermotolerant state are able to reduce activation of SAPK/JNK after heat stress. Notably, the level of activation of SAPK/JNK did not correlate with the amount of apoptosis detected after different stresses. Constitutive HSP72 expression inhibited poly(ADP-ribose) polymerase cleavage in cells after heat shock and TNFalpha but not after ceramide or ionizing radiation. The results suggest either that SAPK/JNK activation is not required for apoptosis in mouse embryo fibroblasts or that HSP72 acts downstream of SAPK/JNK. Furthermore, the data support the concept that caspase activity, which can be down-regulated by HSP72, is a crucial step in stress-induced apoptosis. Based on data presented here and elsewhere, we propose that the heat shock protein family can be classified as a class of anti-apoptotic genes, in addition to the Bcl-2 and inhibitor of apoptosis protein families of genes.