Anti-sense inhibition of small-heat-shock-protein (HSP27) expression in MCF-7 mammary-carcinoma cells induces their spontaneous acquisition of a secretory phenotype.

This work was aimed at testing the hypothesis (hitherto supported only by indirect evidence) that, besides contributing to resistance to stress, the small heat-shock-protein HSP27 might be involved in the control of growth and differentiation in mammary-tumour cells, where it is known to be oestrogen-regulated. Therefore, MCF-7 cells were transfected with a modulatable human hsp27 anti-sense cDNA. Clones of transfectants (designated alphahsp27) were selected which, upon expression of the anti-sense, exhibited a decline in HSP27 accumulation, associated with a decrease in resistance to heat shock and in proliferation rate, the degree of the latter reflecting their respective reduction in HSP27 content. The effects of anti-sense inhibition of HSP27 production were similar to those exerted on parental cells by phorbol myristate (TPA). Both resulted in growth inhibition, accumulation of lipid droplets in the cytoplasm, formation of secretory microvesicles with internal microvilli and increased release of several proteins, including the isoforms of a 52-kDa protein, which we identified as the oestrogen-regulated protein cathepsin D, all this without noticeable change in actin organization. These data constitute the first direct support for the hypothesis that, at least in some cell types, HSP27 might play a modulatory role in cell differentiation and (perhaps by this) in proliferation. While allowing dissociation of this role from the known action of HSP27 on actin polymerization, they suggest similar modulation of the function of some protein(s) implicated in the acquisition of the secretory phenotype by MCF-7 cells, with HSP27 also exerting an inhibitory action that can be alleviated either by its phosphorylation (as occurs with TPA) or by inhibition of its production.

Expression of HSP27 results in increased sensitivity to tumor necrosis factor, etoposide, and H2O2 in an oxidative stress-resistant cell line.

The role of HSP27 in cell growth and resistance to stress was investigated using murine fibrosarcoma L929 cells (normally devoid of constitutively expressed small HSPs) and human osteoblast-like SaOS-2 cells stably transfected with a human hsp27 expression vector. Our data showed that our L929 cells were more resistant to oxidative stress than generally observed for this line. Production of HSP27 in these cells led to a marked decrease in growth rate associated with a series of phenotypical changes, including cell spreading, cellular and nuclear hypertrophy, development of an irregular outline, and a tremendous accumulation of actin stress fibers. By contrast, none of these changes was observable in SaOS-2/hsp27 transfectants overexpressing the protein product. Together, these observations are consistent with a cause-to-effect cascade relationship between increased (or induced) HSP27 expression, changes in cytoskeletal organization, and decreased growth. On the other hand, whereas the transfection of the hsp27 gene increased the cell resistance to heat in both cell lines, only in SaOS-2 cells was this associated with protection to the cytotoxic action of tumor necrosis factor-alpha (TNF-alpha) and etoposide. Unexpectedly, L929/hsp27 transfectants exhibited an increased sensitivity to both agents and also to H2O2. These data thus imply that different mechanisms are involved in the cell resistance to heat shock and to the cytotoxic action of TNF-alpha, etoposide, and H2O2. They also plead against the simple view that overexpression of a phosphorylatable HSP27 would necessarily be beneficial in terms of increased cell resistance to any type of stress. Our data further indicate that the role of HSP27 in cellular resistance to stress and in cell proliferation involves different targets and that the ultimate result of its interference with these processes depends on the intracellular context in which the protein is expressed.

Changes in the phosphorylation status of the 27 kDa heat shock protein (HSP27) associated with the modulation of growth and/or differentiation in MCF-7 cells.

We have used human mammary cells of the MCF-7 strain, which constitutively express high levels of the small heat shock protein HSP27 and we have compared the changes in the phosphorylation status of this protein together with changes in cell growth and/or morphology induced by the action of one of the following agents: (1) TPA (12-O-tetradecanoylphorbol-13-acetate), known as a differentiation inducer in MCF-7 cells; (2) OH-TAM (hydroxytamoxifen), which exerts a cytostatic and cytotoxic action; or (3) TNF alpha (tumour necrosis factor), which induces apoptotic cell death in this cell line. Our data show that TPA and TNF stimulate an immediate and massive phosphorylation of HSP27, whereas OH-TAM affect the phosphorylation status of the protein only after a 3 day delay. In the case of TPA, high levels of HSP27 phosphorylation were maintained for at least 4 days, along with growth inhibition and acquisition by the cells of a secretory phenotype. TPA and OH-TAM exerted similar immediated effects on cell growth, despite the different time course of their action on HSP27 phosphorylation. This excludes the possibility that the latter is a necessary consequence of, or an absolute requisite to, growth inhibition. With OH-TAM and TNF the increase in HSP27 phosphorylation was concomitant with the appearance of apoptosis, not observed with TPA. This indicates that increased phosphorylation of HSP27 is not specifically associated with the triggering or the execution of apoptosis in these cells. Altogether, our data support the concept that phosphorylated HSP27 is involved (and might then be rate limiting in some instances) in the execution of vital cell programmes (including resistance to stress, proliferation and differentiation), as well as in that of cell death. This is consistent with its role in actin polymerization and its position downstream of the p38/RK-type MAPkinase, itself a point of convergence for diverse signal transduction pathways.

Antisense inhibition of the 27 kDa heat shock protein production affects growth rate and cytoskeletal organization in MCF-7 cells.

MCF-7 cells were co-transfected with the human HSP27 antisense cDNA and the neomycin resistance gene, included in the constitutive expression vector pSVL, and the phenotypical changes associated with decreased expression of the HSP27 protein were analysed. Three out of 10 neomycin-resistant clones obtained proliferated normally and showed a normal HSP27 content (Western blot). The seven other clones (designated as alpha HSP27 clones) were characterized by a dramatic growth inhibition associated with alterations in cellular morphology. Cells became progressively hypertrophied, exhibited lamellar protrusions and tended to lose contact with each other. They also acquired characteristics of secretory cells, namely the presence of numerous refractile granules and secretory canaliculi. Among the alpha HSP27 clones, two were immunocytochemically analysed for HSP27 content. Both clones were immunonegative for HSP27, contrary to parental cells and neo-transfectants. Actin immunostaining in one of these HSP27 negative clones revealed that microfilament organization changed from diffuse to punctate distribution. Our data support the current concept of a role for HSP27 in cell growth and differentiation and further suggests that this might occur through a control on actin polymerization-depolymerization.

Antibodies to small ribonucleoprotein and to 73-kD heat shock protein: two distinct markers of mixed connective tissue disease.

We set out to discover whether antibodies to small ribonucleoprotein antigens (RNP) and to 73-kD heat shock protein (hsp 73) which have been proposed as markers of mixed connective tissue disease (MCTD) recognize different epitopes. MCTD serum was immunoadsorbed on hsp 73-coupled Sepharose and the affinity retained, and non-retained fractions were checked by immunoblotting for recognition of either purified bovine hsp 73 or calf thymus extract RNPs. The hsp 73 affinity-bound serum fraction recognized hsp 73 but not RNP antigens, the reverse being true for the non-retained fraction. We conclude that anti-hsp 73 and anti-RNPs are distinct markers of MCTD.

FB1, a monoclonal antibody reacting with a keratin 14 epitope, stains only a small subset of psoriatic basal keratinocytes.

A murine monoclonal antibody, FB1, reacted with the basal keratinocytes of human stratified epithelia. One-dimensional and two-dimensional immunoblotting assays, performed on keratins extracted from HaCat cells and normal human keratinocytes, showed that FB1 recognizes K14. When LL002, another K14 monoclonal antibody is added, the FB1 stained area in the 2D-immunoblot seems to cover a fraction of the LL002 spot. Immunohistochemical data obtained from studies on normal human tissues supported the K14 specificity of FB1, but when compared with two other monoclonal antibodies, LL002 and RCK107 reacting with K14, some differences appeared. These differences were mainly seen in sweat glands, hair follicles, psoriatic epidermis and salivary glands. In psoriatic epidermis, FB1 showed a heterogeneous pattern of staining of the basal cell compartment. Intense reactivity was only observed at the bottom of the rete ridges. Staining diminished and finally disappeared in the basal cells above the dermal papillae. This observation supports the view that an increased germinative cell population in psoriasis involves a partially differentiated amplifying compartment in which the number of cell divisions is increased.

Antibodies to the constitutive 73-kd heat shock protein: a new marker of mixed connective tissue disease?

PURPOSE, PATIENTS, AND METHODS: This report documents the finding of an elevated titer of IgG reacting with the constitutive bovine 73-kd heat shock protein (HSP) in the serum of patients with rheumatoid arthritis, scleroderma, and mixed connective tissue disease (MCTD). RESULTS AND CONCLUSIONS: Further characterization of antibodies from patients with MCTD showed that the antibodies also recognize the human constitutive 73-kd HSP, but not the inducible 72-kd isoform. Very high levels of antibodies appeared to be specific for MCTD; the differences between levels in patients with MCTD and those in healthy subjects (blood donors) were highly significant (p < 10(-8)), with no values in this group of patients overlapping with those in the controls. This parameter might therefore represent a new diagnostic marker for this disease.

Expression of 27-kD heat-shock protein isoforms in human neoplastic and nonneoplastic liver tissues.

Previous study of rat liver during chemically induced hepatocarcinogenesis has shown that expression of isoforms of the 27-kD heat-shock protein was greater in neoplastic nodules and in hepatocellular carcinoma than in control livers. In this study, various human neoplastic and nonneoplastic liver tissues were investigated with electrophoresis after amino acid labeling to evaluate the expression of 27-kD heat-shock protein isoforms. This revealed that human liver contains 27-kD proteins that are recognized by a polyclonal antibody raised against human 27-kD heat-shock protein. Basal levels of fluorographical and immunostaining intensity of the 27-kD heat-shock protein spots (respectively, after [3H]leucine or 32P incorporation or as checked with a specific human 27-kD heat-shock protein antibody) were higher in hepatomas than in non-tumorous liver. Phosphorylation patterns of the 27-kD heat-shock protein isoforms were, however, similar in hepatocellular carcinoma and in uninvolved surrounding liver. Heat inducibility of the 27-kD heat-shock protein, tested in one case of liver cell adenoma and in the surrounding liver, was also preserved in both tissues. The role of the overexpression of 27-kD heat-shock protein in neoplastic liver tissues remains unknown. We propose, as a working hypothesis, that it is related to the resistant phenotype acquired by some tumors during malignant progression.

Enhanced expression of a 27 kD protein during diethylnitrosamine-induced hepatocarcinogenesis in rats.

Patterns of neosynthesized cellular proteins from normal rat liver and from diethylnitrosamine-induced neoplastic nodules and hepatocellular carcinomas were analyzed by radiolabeling and fluorography of two-dimensional gel electrophoregrams. Three proteins exhibited a significant and reproducible increase in labeling intensity in the nodules (n = 5) and in the tumors (n = 10) as compared to the normal liver (n = 10). Two of those proteins (MW 31 and 33 kD, pI of 5.25, 5.15 respectively) are secreted proteins and as yet, we have no clue as to their nature. The third one is an intracellular protein of 27 kD pI 5.5. Several similarities in physico-chemical properties (MW, pI, phosphorylated state, low methionine content) indicate that this 27 kD protein might be the 27 kD heat shock protein (27 HSP). This is further supported by our observation that the cadmium-induced 27 HSP comigrates with our 27 kD protein.

Effect of aggregated immunoglobulins on mononuclear cell protein production in rheumatoid arthritis.

The synthesis and secretion of proteins, especially interleukin 1 (IL-1), by mononuclear cells from patients with rheumatoid arthritis (RA) in response to heat-aggregated IgG (HAGG) has been investigated. Mononuclear cells were labeled with 35S-methionine during 1 hr of HAGG stimulation, and the intracellular and extracellular protein content was measured and compared by fluorography. HAGG dramatically accelerated protein release into the medium. This was observed in both normal and RA mononuclear cells but was much more marked with the latter. The phenomenon was correlated with the release of a IL-1 activity measurable in the thymocyte proliferation assay with a more pronounced labeling in the extracellular medium of a 14-kDa band not detectable in the intracellular pattern. Partially purified proteins between 14-20 kDa recovered by transblotting also stimulated thymocyte proliferation. These results therefore suggest a particular sensitivity of RA mononuclear cells to HAGG, resulting in a very rapid de novo synthesis and an accelerated secretion rate of IL-1.

Effect of aggregated IgG on mononuclear and polymorphonuclear cell cooperation.

The leukocyte adherence inhibition (LAI) test was used to determine the response of different leukocyte subpopulations in rheumatoid arthritis (RA) patients and normal donors to heat-aggregated gammaglobulins (HAGGs). The adherence of polymorphonuclear cells of (PMNs) could be inhibited either directly by high concentrations of HAGGs or indirectly by soluble factors secreted by RA mononuclear cells incubated with low amounts of HAGGs. The stimulatory site of the IgG was located on the Fc fragment. Since the LAI reaction was also observed with material recovered from rheumatoid synovial fluid, tissue, and nodules, these results could have clinical relevance in RA.

Characterization of gamma-glutamyltransferase from neoplastic and non-neoplastic liver tissues in man and during rat liver hepatocarcinogenesis.

The activity and the affinity for concanavalin A-Sepharose (Con A) of liver gamma-glutamyltransferase (gamma GT) were investigated in man, under various clinical conditions and in rats during experimental hepatocarcinogenesis. In man, gamma GT activity was higher than normal in hepatomas and (except for 1 case of hemochromatosis) also higher in the surrounding cirrhotic liver. The proportion of gamma GT which did not bind to Con A (Con A- form) was also increased in the tumors and in the surrounding liver, yet (with the same exception as above) to a greater extent in the hepatomas. In rat, gamma GT activity was higher in fetal liver (15-fold) and in hepatocarcinomas (10-fold) than in normal adult liver; total liver gamma GT activity gradually increased during progression from foci of altered cells to neoplastic nodules and tumors. The proportion of the Con A- form of gamma GT in the early or late stage of the carcinogenic process did not significantly differ from that in normal adult or regenerating rat liver, i.e. about 20% of the total activity. By contrast, nearly all the gamma GT from fetal rat liver bound to Con A. This suggests that gamma GT expression in rat liver carcinoma does not correspond to so-called retrodifferentiation process.

Partial characterization of the soluble mediator of leukocyte adherence inhibition.

The leukocyte adherence inhibition (LAI) test in arthritis is a model of cooperation between monocytes, T cells and polymorphonuclear cells (PMN). Mononuclear cells (MNC) from patients with rheumatoid arthritis in contact with aggregated IgG release within 60 min in the test tube a supernatant capable of inhibiting the adherence of PMN to glass (LAI activity). By protein labeling, fractionation and electroblotting, we showed that the supernatant LAI activity was located in the 14-20 kDa region of the gel and was actively secreted upon MNC stimulation. Under negative conditions, the factors with LAI activity were able to constitute larger complexes and their charge was heterogeneous. Their relationship to interleukin-1 (IL-1) is discussed, since the supernatant gave a positive response in the thymocyte proliferation assay; small amounts of IL-1 had the capacity to inhibit PMN adhesiveness and anti-IL-1 blocked this activity.

Estrogen-induced protein in the rat uterus: association with actin.

The affinity of the estrogen-induced protein, IP, for reconstituted F-actin in uterine homogenates was investigated. The results demonstrate that a significant proportion of a 46 K protein, of which the rate of synthesis is increased by estrogen, two properties of BB-CK, co-sedimented with in vitro re-polymerized actin.