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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Differential blockade of estrogen-induced uterine responses by the antiestrogen nafoxidine.

We have used an experimental design described by Gardner et al. [6] for dissociating early and late uterine responses to estradiol, involving pretreatment of immature rats with 5 micrograms nafoxidine (Upjohn U-11, 100 A, UA) for 24 h, before administrating estradiol. In these conditions the authors showed that responses occurring 4-h after estradiol administration were not blocked, while 24-h responses were abolished.

Estrogen-induced proteins in luminal epithelium, endometrial stroma and myometrium of the rat uterus.

The early effect of estrogen on the synthesis of cytosolic proteins was investigated in the luminal epithelium, endometrial stroma and myometrium of the uterus in adult ovariectomized rats. The procedure of Reiss and Kaye (1981) was followed (involving two-step fractionation of 35S-labelled proteins and fluorographic analysis) except that the uteri were fractionated into their three main tissue components before homogenization. The results show that E2 stimulates the synthesis of BB-CK (brain-type creatine kinase), the major component of IP (estrogen-induced protein), in the three tissues.

Nafoxidine-responsive uterine protein: further characterization and distinction from the "estrogen-induced" protein (IP).

In a previous paper, we reported that nafoxidine (UA) stimulated the synthesis of a uterine protein showing the same electrophoretic mobility as the "estrogen-induced protein" (E2-IP) first described by Notides and Gorski. In the present work, we analyzed the IP-containing electrophoretic zone by SDS polyacrylamide-gel electrophoresis, and found that estradiol-17 beta (E2) and nafoxidine stimulated the synthesis of different proteins. As expected, estradiol-17 beta stimulated the synthesis of the E2-IP of 46 000 Mr.