Evidence for a role of FGF-2 and FGF receptors in the proliferation of non-small cell lung cancer cells.

Basic fibroblast growth factor (FGF-2) has been implicated in the progression of human tumours via both autocrine and paracrine (angiogenic) activities. We investigated the expression of FGF-2 and FGF receptors (FGFR-1 to -4) in NSCLC cell lines (N = 16), NSCLC surgical specimens (N = 21) and 2 control cell lines. Our data show that almost all NSCLC cells produce elevated levels of FGF-2 and FGFR in vitro and in vivo. FGF-2 expression did correlate with a short doubling time as well as with potent anchorage-independent growth of NSCLC cell lines. In contrast with control cells, NSCLC cells did not secrete considerable amounts of FGF-2 into the extracellular space. Expression levels of FGFR-1 and -2 in NSCLC cell lines correlated with FGF-2 production. FGFR were located at the plasma membranes in some low FGF-2-producing NSCLC and control cell lines. These cells were sensitive to the proliferative effect of recombinant FGF-2 (rFGF-2). In NSCLC cell lines with an enhanced FGF-2 production, representing the majority studied, FGFR localisation was predominantly intracellular. These cells were insensitive to both the proliferative effect of rFGF-2 and growth inhibition by FGF-2-neutralising antibodies. In contrast, several agents antagonised FGF-2 intracellularly impaired growth of almost all NSCLC cell lines. Our data suggest a role of FGF-2 and FGFR in the growth stimulation of NSCLC cells possibly via an intracrine mechanism.

Expression of the 72-kD heat shock protein is induced by ultraviolet A radiation in a human fibrosarcoma cell line.

The 72-kD heat shock protein (hsp72) belongs to a family of stress inducible proteins (heat shock proteins, hsp) and its expression is associated with increased survival of cells in culture following exposure to ultraviolet radiation (UV). Hsp72 can be induced by a number of stresses, including heat, cold, and toxic chemicals. The purpose of this study was to evaluate whether UV is able to activate transcription of hsp72. The human fibrosarcoma cell line HT1080 was used for these experiments because hsp72 is not detectable in these cells under normal culture conditions. Cells were exposed to UVA and UVB using a solar simulating source and hsp72 was determined in whole cell extracts by immunoblotting. For inhibition of mRNA and protein synthesis cordycepin (20 microg/ml) and cycloheximide (10 microg/ml) were added to the cultures, respectively. UVA-induced lipid peroxidation was inhibited by alpha-tocopherol and butylated hydroxytoluene (BHT). UVA but not UVB induced hsp72 with maximal expression at 40 J/cm2, 8-12 h after exposure. Induction was blocked by cordycepin as well as by cycloheximide indicating that both, mRNA and protein synthesis, are required for UVA-induction of hsp72. Inhibition of cell lipid peroxidation with alpha-tocopherol and BHT had no effect on hsp72 expression. These results suggest that induction of hsp72 is part of an adaptive response mechanism in human cells to UV-related stress.

Expression of the small heat shock protein HSP 27 in developing human skin.

The 27 kDa heat shock protein (HSP 27) is expressed in keratinocytes of the upper epidermal layers, and recent evidence suggests that this protein is involved in the regulation of epidermal differentiation. The expression of HSP 27 was investigated in developing human skin by immunohistochemistry utilizing a specific monoclonal antibody. We used formalin-fixed, paraffin-embedded tissue of abdominal skin obtained from 34 human fetuses ranging between 13 and 30 weeks estimated gestational age (EGA). We found that HSP 27 is not expressed in keratinocytes until week 14 EGA. At this stage staining is observed in the periderm and the upper intermediate cells but not in hair germs. During further development, HSP 27 expression correlates with increasing epidermal differentiation, i.e. shedding of the periderm and beginning of keratinization. HSP 27 expression is confined to the upper cell layers and sparse basal cells. In hair follicles, HSP 27 can be detected in the innermost cell layer of the outer root sheath and in keratinocytes of the bulge identical to what is observed in adult skin. The hair papilla, matrix cells and sebaceous glands are negative for HSP 27 and remain so during further development. In eccrine sweat glands of the 24th week EGA, HSP 27 is confined to the superficial cell layer of the sweat ducts. In the present report we demonstrate differentiation-related expression of HSP 27 in developing human skin. Further in vitro studies will address the molecular function of HSP 27 in epidermal differentiation and development.

Modification of growth in small heat shock (hsp27) gene transfected breast carcinoma.

The role of hsp27 in the regulation of cell growth has been investigated in the breast cancer cell line MDA-MB-23 1. Cells were co-transfected with an expression vector carrying the human hsp27 gene (pSG-2711) and a plasmid conferring neomycin resistance (pWlneo). Transfected cells were selected for neomycin resistance. Stable transfectants were used as a pooled population for further experiments, since single-cell colonies were not able to grow into mass culture under continuous selection pressure. Over-expression of hsp27 was analysed by immunohistochemistry and immunoblotting. Cells only transfected for neomycin were used as control cells. The growth rate of the transfected cell line was determined whether overexpression of hsp27 directly influences the growth properties of the cells. Growth analysis of transfected cell lines in vitro revealed a lower proliferation rate of the hsp27 overexpressing cells compared to controls. These data suggest that hsp27 is involved in downregulation of cell proliferation in this human breast cancer cell line.

Overexpression of the small heat shock protein, hsp27, confers resistance to hyperthermia, but not to oxidative stress and UV-induced cell death, in a stably transfected squamous cell carcinoma cell line.

The 27 kD heat shock protein (hsp27) is expressed in human keratinocytes in association with differentiation in vitro and in situ. This study was conducted to investigate whether the expression of hsp27 in keratinocytes is associated with increased resistance to the deleterious effects of heat and UV radiation. A transfection vector carrying the human gene for hsp27, under the control of hsp27 as well as the SV40 promoter (pSG2711, M. Jäättelä et al., EMBO J. 11 (1992) 3507-3512), was introduced together with a neomycin-resistance gene into the squamous cell carcinoma cell line A431. Cells were exposed to either UVA, UVB, head (45 degrees C, 4 h) or hydrogen peroxide (0.025-0.5 mM) and the percentage of surviving cells was determined. Overexpression of hsp27 induced increased resistance to hyperthermia, but not to hydrogen peroxide-mediated oxidative injury. When cells were exposed to increasing amounts of UVA (5-80 J cm-2) and UVB (4-64 mJ cm-2), the percentage of surviving cells was identical for clones overexpressing hsp27 and control clones. From these data, we conclude that hsp27 is a mediator of thermotolerance, but does not protect keratinocytes from UV-induced cell death.

Stress proteins in the cellular response to ultraviolet radiation.

Virtually all cells-from prokaryotes to highly differentiated mammalian tissues-respond to a sudden increase in temperature with increased production of a limited set of proteins, called heat shock proteins or stress proteins (hsp). Other stress factors such as alcohol, heavy metals, oxidants and agents leading to protein denaturation are equally able to induce a similar response. Induction of hsp is followed by a transient state of increased resistance to further stress. Many hsp function as "molecular chaperones" by binding to partially folded or misfolded proteins thus preventing their irreversible denaturation during stress exposure. The high evolutionary conservation of this reaction suggests its importance for the survival of cells and tissues under hostile environment conditions. Ultraviolet radiation (UV) exerts many potentially harmful effects on prokaryotic and eukaryotic cells and hsp may help the cell to cope with UV-induced damage. This review will focus on the role of hsp in the cellular response of mammalian skin to UV. Hsp have been detected in resting as well as stress exposed epidermal and dermal cells and experimental evidence points to the fact that these proteins mediate protection from UV induced cell death in vitro and in vivo. Experimental studies further indicate that UV itself might be able to induce the expression of specific hsp. Thus, hsp might provide an adaptive cellular response to increasing exposure to UV. Furthermore, UV-activation of hsp synthesis may provide a valuable model for investigation of the transcription regulation of UV-induced gene expression.

Modification of growth and tumorigenicity in epidermal cell lines by DNA-mediated gene transfer of M(r) 27,000 heat shock protein (hsp27).

In the present communication, the role of the M(r) 27,000 human small heat shock protein (hsp27) in tumorigenicity was examined. Stable transfectants of a melanoma cell line (A375) and an epidermal squamous carcinoma cell line (A431), isolated by cotransfection of a hsp27 expression vector (pSG-2711) and a neomycin-resistant plasmid, were obtained. Clones expressing high levels of hsp27 were analyzed using immunohistochemistry and immunoblotting. Cells transfected with only the plasmid for neomycin were used as control cells. Growth analysis of transfectants in A375 and A431 tumor cells showed in vitro a lower proliferation rate than control clones derived from both lines. To investigate the correlation of hsp27 expression and tumorigenicity, transfectants of each cell type and control cells were injected into nude mice. A delay in tumor development was detected in animals inoculated with cells overexpressing hsp27. However, after this initial delay, tumors appeared in some of these animals and no difference could be observed in their growth dynamics compared to control tumors. When tumors transfected with the hsp27 construct were analyzed using immunohistochemistry and PCR, no evidence for hsp27 expression was obtained which implicates instability of the transduced foreign DNA when maintained under nonselective conditions. The present study shows that genetic manipulation of tumor cells may provide valuable information on the role of hsp27 in tumor growth.

Alveolar macrophages of patients with adult respiratory distress syndrome express high levels of heat shock protein 72 mRNA.

Adult respiratory distress syndrome (ARDS), a multifactorial disease with poor prognosis, is characterized by an accumulation of inflammatory cells within the airspaces of the lungs. There is evidence that alveolar macrophages (AM) are involved in the pathogenesis of this pulmonary disease. It has been demonstrated that AM synthesize heat shock proteins (HSPs) after exposure to certain stress factors. Increasing evidence suggests that HSPs could confer protection against oxidative injury, noxious molecules, and bacterial toxins. In stressed cells HSP 72 appears to be essential for survival during and after exposure to cellular injury. The aim of this study was to evaluate the magnitude of HSP 72 expression by human AM of patients with ARDS and correlate that with respiratory burst activity. Bronchoalveolar lavage was performed in six ARDS patients, 10 patients with high risk for developing ARDS, and two patients who underwent bronchoscopy for other reasons. Spontaneous ex vivo expression of HSP 72 in AM could be demonstrated by immunocytochemistry. Total RNA as well as poly(A)-rich mRNA were extracted from recovered AM and analyzed by Northern blot and slot blot using a human HSP 72-specific probe. Signals of slot blot were analyzed by densitometry and expressed as relative levels of HSP 72 mRNA of stressed (42 degrees C) HT 1080 control cells. Significantly (p < .001) higher levels of HSP 72 mRNA were measured in patients with ARDS (96.2 +/- 9.5 relative levels) in comparison to those not developing this syndrome (46.0 +/- 4.2). With regard to respiratory burst activity of AM in patients with ARDS, there was a negative correlation between HSP 72 expression and reactive oxygen species production. The AM of patients with ARDS with high relative levels of HSP 72 expression showed low respiratory burst activity. A predictive value for disease severity of high level of HSP 72 mRNA in AM in patients at risk for ARDS has to be evaluated by future studies. This demonstration of HSP 72 expression ex vivo suggests a protective role of HSP response against endo/exogenously generated stress factors in AM.

Expression of the 27-kDa heat shock protein in human epidermis and in epidermal neoplasms: an immunohistological study.

The 27-kDa heat shock protein (HSP27) is a member of the small heat shock protein (HSP) family. In addition to its putative function in thermotolerance, this protein may play a part in the regulation of cell growth and differentiation. This study was conducted to assess the significance of the expression of HSP27 in human epidermis and in cutaneous neoplasms. Sixty-two biopsy samples from normal human skin and from inflammatory and neoplastic skin diseases were investigated by immunohistochemistry on formalin-fixed paraffin-embedded tissue sections, using a monoclonal antibody specific for HSP27. In normal human epidermis, HSP27 is expressed in the upper epidermal layers with a cytoplasmic staining pattern. The basal cell layer does not express detectable amounts of HSP27. In hair follicles, staining is mainly confined to the outer root sheath and to the infundibular epithelium. Melanocytes, dermal fibroblasts and endothelial cells do not express detectable amounts of HSP27. HSP27 could not be detected in fetal skin until the 20th week of gestation. Tumour cells in basal and squamous cell carcinomas do not express significant amounts of HSP27. In solar keratoses, seborrhoeic keratoses, human papillomavirus (HPV)-induced hyperproliferative lesions and inflammatory skin conditions, HSP27 expression largely resembles the pattern observed in normal human skin. HSP27 is expressed in a differentiation-related pattern in normal human epidermis and hyperproliferative disorders of the epidermis. We conclude that HSP27 may be regarded as a marker of differentiation in epidermal keratinocytes. Absence of HSP27 in the upper epidermal layers may be a marker for epidermal malignancy.

72-kD heat shock protein is a mediator of resistance to ultraviolet B light.

We have shown previously that human epidermal keratinocytes in situ and in vitro constitutively express high levels of the 72-kD heat shock protein (hsp72) and that hsp72 expression in these cells can be further induced with heat treatment. In the present study, we continue our investigation of the ultraviolet (UV) B protective effect of hyperthermic treatment and ask whether hsp72 is a mediator of heat-shock-induced UVB resistance. The results of our experiments demonstrate that heat treatment (42 degrees C for 4 h) before UVB exposure is able to increase significantly the UVB resistance of the epidermal carcinoma cell line A431. Heat-induced UVB resistance was most pronounced if the cells were exposed to UVB immediately after heat treatment. The protective effect was not detectable beyond a recovery period of 12 h. To investigate the role of hsp72 in hyperthermia-induced UVB resistance, we inhibited the expression of this protein using either a specific antisense oligodeoxynucleotide or quercetin, a flavonoid that has been shown to down-regulate hsp expression. Treatment with the oligomer as well as with quercetin significantly increased the susceptibility of A431 to UVB-induced damage and nullified the protective effect of heat preconditioning. A noncomplementary control oligodeoxynucleotide had no significant effect. These results indicate that heat treatment is able to induce a state of increased resistance to the deleterious effects of UVB in human keratinocytes in vitro. hsp72 is a molecular mediator of this protective effect, and its constitutive expression in human epidermal keratinocytes may be an important mechanism for the protection of human epidermis from UVB-induced damage.

Increased expression of the M(r) 27,000 heat shock protein (hsp27) in in vitro differentiated normal human keratinocytes.

The M(r) 27,000 heat shock protein (hsp27) is a member of the small heat shock protein family. Cell differentiation is a process in which a role for small heat shock proteins has been suggested. The ability to control the state of differentiation in normal human keratinocytes by modification of extracellular calcium concentration makes it an ideal in vitro system for exploration of the specific components and steps in differentiation. We have focused on the in vitro expression of hsp27 in undifferentiated and differentiated human normal keratinocytes (HNK) as a marker for differentiation. Immunological methods (immunohistochemistry and immunoblotting) as well as Northern blotting were used. Cells of the breast cancer line MCF-7 served as a positive control. We demonstrated that hsp27 was expressed at low levels in normal human keratinocytes, kept under calcium concentrations where cells formed discrete colonies of undifferentiated, noncornified cuboidal cells (0.03 mM Ca2+), and linked cuboidal cells with a noncornified appearance (0.15 mM Ca2+). Upon cultivation in high calcium (1.00 mM Ca2+) where a more morphological state of differentiation was reached, more spindle shaped with cornification of individual cells, a 2-fold increase in hsp27 expression was observed. A somewhat weaker increase in hsp27 mRNA was shown by Northern blot analysis. Our studies provide evidence that hsp27 is accumulated in a differentiation-dependent manner in human normal keratinocytes grown under conditions inducing terminal differentiation (0.03-1.00 mM Ca2+). Therefore, hsp27 can be regarded as a marker of differentiation in human normal keratinocytes.

Human keratinocytes in vivo and in vitro constitutively express the 72-kD heat shock protein.

Exposure of cells to elevated temperatures induces a physiologic response characterized by the synthesis of a specific set of proteins (heat shock or stress proteins, HSPs) mediating repair mechanisms and protection from cellular damage. In the present study upon immunohistochemistry using a specific monoclonal antibody, the constitutive and heat-induced expression of the 72-kD HSP (HSP72) in normal human skin and in human epidermal cell lines (KB, A431) was investigated. Normal (unstressed) epidermis and adnexal structures of normal human skin were found to constitutively express HSP72. In contrast, a substantial HSP72 expression could not be observed in the dermal cellular compartment. In vitro heat treatment of punch biopsies from normal skin (42 degrees C, 4 h) resulted in a further increase of epidermal HSP72 expression. In addition, dermal cells were found to be induced to express HSP72. To further evaluate the spontaneous HSP72 expression of epidermal cells two epidermoid carcinoma cell lines (A431, KB) were investigated. Upon immunohistochemistry and Western blot analysis a significant HSP72 expression could be detected in unstressed KB and A431 cells. In contrast, a human fibrosarcoma cell line (HT1080) was negative for HSP72 at 37 degrees C but upon heat treatment a strong induction was observed. Furthermore, Northern blot analysis using a cDNA probe specific for human HSP72 revealed a constitutive expression of HSP72 mRNA in both epidermal cell lines. These findings demonstrate a significant expression of the stress-inducible HSP72 in unstressed human skin as well as in epidermal cell lines, suggesting that HSP72 may inherently be involved in the protective function of normal human skin.

Granulocytes of critically ill patients spontaneously express the 72 kD heat shock protein.

Polymorphonuclear neutrophils (PMN), through their ability to release oxygen-free radicals and other tissue-damaging molecules, play a major role in the pathogenesis of multiple organ failure syndrome (MOFS). There is evidence that heat shock proteins (stress proteins; HSPs) are involved in cellular repair mechanisms, and are protecting cells against oxidative injury. In this study, we analyzed the spontaneous expression of the 72 kD HSP (HSP72) in peripheral blood PMN of 20 critically ill patients (16 polytrauma victims, four patients after major surgery) admitted to an intensive care unit. The expression of HSP72 was investigated in PMN of patients and healthy donors by immunohistochemistry. We found spontaneous expression of HSP72 in PMN of 12 (60%) of 20 patients. No specific staining was detected in PMN of healthy donors (n = 10). In PMN of six of 12 patients expressing HSP72 without previous heat treatment, we found an impairment of respiratory burst activity (RBA) compared to the control population. These results demonstrate for the first time the in vivo expression of HSP72 in human leukocytes without previous heat treatment, and suggest a possible role of this protein in patients suffering from severe tissue injury.

Similar frequency of autoantibodies against 70-kD class heat-shock proteins in healthy subjects and systemic lupus erythematosus patients.

Stress or heat-shock proteins may be involved in the initiation and perpetuation of autoimmune diseases. In order to investigate a possible role of autoantibodies against the 70-kD family of heat-shock proteins in systemic lupus erythematosus (SLE), sera of SLE patients and healthy subjects were tested for the presence of IgG and IgM antibodies to 70-kD class proteins. These proteins were purified by affinity chromatography on ATP-agarose and used in Western blotting studies. The data obtained revealed that antibodies to the 72-kD and the 73-kD heat-shock proteins occurred with similar frequencies both in healthy subjects and SLE patients. Thus, approximately 20% of the sera in each group contained IgG antibodies, and IgM antibodies were detected in about 30% of the sera tested. Moreover, in SLE patients no association between the occurrence and titre of these antibodies and disease activity was found. These data suggest that antibodies to the 70-kD class heat-shock proteins are naturally occurring and argue therefore against an involvement of these antibodies in the pathogenesis of SLE.

Human autoantibodies against a nucleolar protein.

Autoimmune sera showing prominent immunofluorescence in nucleolus were selected and analysed by immunoblotting techniques. Immunoblots using a nucleolar extract as antigen source revealed sera recognizing a 38 kDa nucleolar protein. Low concentration of Actinomycin D, which inhibits the ribosomal RNA synthesis, caused a loss of fluorescence. This suggests that the nucleolar antigen may be associated with the assembly of packaging of the ribosomes. The present nucleolar antigen has properties similar to the previously described nucleolar phosphoprotein B23 of rat cells and the recently described nucleolar protein NO38 of mouse and Xenopus cells.

Identification of a yeast ribonuclease H as an Sm antigen.

We have isolated a 55-kDa enzyme from Saccharomyces cerevisiae on the basis of its ability to hydrolyze specifically the RNA moiety of RNA/DNA hybrids [RNase H(55)]. Remarkably, monospecific anti-[RNase H(55)] antibodies revealed that the protein associates with several small RNAs, including some of the essential yeast spliceosomal snRNAs. Moreover, immunoprecipitation as well as immunoblotting experiments demonstrated that the yeast enzyme reacts (a) with human anti-Sm autoantisera, (b) with a monoclonal antibody specific for the human snRNP proteins B/B', but (c) not with U1-ribonucleoprotein-specific autoantibodies. These results disclosed a hitherto unexpected degree of evolutionary conservation in snRNP protein structure between yeast and man. Additionally, our findings suggested a re-evaluation of the enzymatic mechanism of RNases H which recognize both RNA and RNA/DNA hybrids.

Nuclear antigens recognized by antibodies present in liver disease sera.

Nuclear and nuclear matrix proteins of HeLa cells were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and subsequently transferred onto nitrocellulose. Antibodies present in sera of patients with primary biliary cirrhosis and autoimmune chronic active hepatitis reacted with some of the blotted proteins. The antibodies were mainly directed against chromatin-associated proteins and protein constituents of discrete RNP particles. In addition, antibodies found in autoimmune liver disease sera detected a hitherto undescribed nuclear protein of 54 kD, and a nuclear matrix protein of approximately 150 kD. Antibodies recognizing a nuclear 25 kD doublet apparently constituted a marker antibody for autoimmune liver disease. Those directed at the 17 kD centromere protein were associated with the primary biliary cirrhosis-related CREST syndrome, while those recognizing La antigen were related to cases of sicca syndrome associated with autoimmune liver diseases.

Transport of beta-globin mRNA from nuclei of murine Friend erythroleukemia cells. Reversible inhibition of transport by the oxidizing sulfhydryl reagent o-iodosobenzoate.

An in vitro assay system for analysis of beta-globin mRNA transport is described. Nuclei isolated from murine Friend erythroleukemia cells induced to synthesize globin mRNA, were incubated in micro-assays. By electrophoresis and hybridization analysis, released 9-S beta-globin mRNA was shown to be undegraded. After direct blotting, the released mRNA was quantified by hybridization with a labeled plasmid containing a beta-globin DNA restriction fragment. The inducibility of beta-globin mRNA transport corresponded to that previously reported for the release of rapidly labeled RNA in other assay systems. In contrast to the ineffectiveness of high concentrations of the sulfhydryl reagent iodoacetate, low concentrations of the oxidizing sulfhydryl reagent, o-iodosobenzoate, inhibited the release of beta-globin mRNA from nuclei of erythroleukemia cells, as well as the release of rapidly labeled RNA from rat liver nuclei. The inhibitory effect of the oxidizing agent on beta-globin mRNA transport could be reversed by postincubation of the nuclei with the reducing agent, dithiothreitol. The potential role of disulfide bond formation on RNA transport is discussed.