Nicole Mairesse

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 αHSP27 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 αHSP27 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.

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 αhsp27) 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. Int. J. Cancer 82:574–582, 1999.

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‐α) 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‐α, 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. J Cell Physiol 177:606–617, 1998.

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. This suggests that BB-CK is related to a function that is common to the estrogen responses (such as hypertrophy) of all three uterine tissues in ovariectomized adult animals. The synthesis of two unidentified proteins of 37000 and 27000 Mr was markedly stimulated in the epithelium. These proteins are probably rate-limiting in responses to estrogen treatment that are specific to the epithelium. The 27000 Mr protein has the same charge as that of the 27000 Mr nafoxidine-induced protein described previously (Mairesse et al., 1981) and is probably therefore the same protein.

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) TNFα (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.

Relationship between the estrogen induced protein IP and other parameters of estrogenic stimulation. A hypothesis

Abstract Data concerning the estrogen-induced protein “IP” discovered by Notides and Gorski are reviewed. Estrogen specificity of the IP response, physiological occurrence of IP and inducibility of an IP-like protein in target tissues other than the rat uterus are considered. Available information concerning the relative ability of different estrogenic and anti-estrogenic compounds to promote IP synthesis, the uterotrophic responses and the receptor depletion-replenishment cycle is examined. From this the hypothesis is proposed that IP induction may be related with receptor replenishment. Recent data concerning the “receptor cycle” are briefly reviewed in order to envisage further extension of our hypothesis.

Estrogen-induced protein in the human breast cancer cell line MCF7

Summary Present data show that a cytosolic protein, similar in charge and molecular weight to the uterine IP, is constitutively synthesized in two mammary tumor cell lines containing (MCF-7) or lacking (Evsa-T) the estrogen receptor (ER). Estrogen treatment stimulates amino-acid incorporation in this protein in the estrogen responsive line MCF-7, only. Further work is needed before concluding about the identity of this 46000 dalton protein and the uterine IP.

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. These findings were defined and extended in the present investigation which shows that: (1) The overall wet weight response of the uterus to estradiol in UA-pretreated animals is decreased when compared to saline pretreated rats. (2) The early increase in cGMP content induced at 2-4 h by estrogen is also decreased but not abolished by the pretreatment with UA, contrary to the late increase in cGMP, which is abolished. (3) The late estrogen-induced proliferative response, measured by the [3H]thymidine labeling index, in the myometrium, stroma and luminal epithelium is maintained after pretreatment with UA. It is remarkable that this occurs in the absence of any estrogen induced uterine hypertrophy as measured by the 24-h increase in uterine weight and RNA or protein content. These results strongly support the hypothesis proposed by Gardner et al. [6] that different control mechanisms might regulate early and late uterine responses to estrogen. Our data suggest the existence of the following dissociable groups of response: (1) Wet weight increase and early increase in cGMP content, (2) Late hypertrophy and second rise in cGMP content and (3) Proliferative response; which are respectively, moderately depressed, abolished or unaffected by UA pretreatment.

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.

Estrogen-induced synthesis and secretion of proteins in the human breast cancer cell line MCF-7.

Abstract The estrogen-induced tumor protein previously described by us in the receptor-positive MCF-7 cells was resolved here by two-step fractionation into four proteins of 46, 5Z 54 and 60,000 Mr. These proteins differ from the uterine IP (48,000 Mr) by being slightly more acidic and by lacking creatine kinase activity. Furthermore, they are not stimulated in Evsa-T cells lacking the estrogen receptors. In MCF-7 cells estrogenic stimulation also provokes a broad spectrum of changes in the secreted proteins. Some proteins (of 37, 46, 54 and 60,000 Mr) are synthetised and secreted at higher than basal rate. A preexisting protein of low turn-over rate (50,000 Mr) is secreted at higher rate after E 2 treatment. In view of these observations, it seems obvious that the examination of a sole protein as a criterion of hormone-dependence should be misleading.