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The membrane type-1 matrix metalloproteinase (MT1-MMP), a protease originally identified in breast carcinoma, is characterized by its capacity to activate other MMPs (MMP-2 and MMP-13) and to degrade extracellular matrix. Our study was undertaken to localize and identify the MT1-MMP expressing cells in human breast adenocarcinomas. A textural analysis of images obtained by immunohistochemistry and in situ hybridization showed precisely the co-expression of alpha smooth muscle actin (alphaSM actin) and MT1-MMP in myofibroblasts. MT1-MMP expression is confined to myofibroblasts in close contact with tumor cells. In sharp contrast, the expression of MMP-2 was more widely distributed in both alphaSM actin positive and negative cells close to and at distance from cancer cell clusters. Our in vitro observations are consistent with the higher level of MT1-MMP expression and of MMP-2 activation observed in alphaSM actin positive fibroblasts derived from breast tumors, as compared to normal breast fibroblasts. Collectively, these results implicate myofibroblasts as major producer of MT1-MMP in breast cancer and emphasize the importance of stromal-epithelial cell interactions in their progression., (Copyright 2003 Wiley-Liss, Inc.)

Klinefelter's syndrome (KS) concerns men and is usually characterized by tallness, underdeveloped testes and sterility. It is generally due to the 47,XXY genotype, ie one extra X chromosome in each cell. Its estimated frequency among newborn boys is 1/500 to 1/700. It seems that 64% KS would be undiagnosed. Abnormally low levels of testosterone blood values are very common in this syndrome. In this case, replacement androgen therapy should be initiated (ideally at the age of 11-15) which prevents osteoporosis and enhances secondary sexual features. Case report - Since early childhood, Mr X has been shy, passive with few friends. When he was 13 years old, the school physician noted a delay of puberty and referred him to an endocrinologist who diagnosed KS. Androgen therapy was introduced but rapidly stopped, because the boy and his parents thought it was useless. Mr X consulted a psychiatrist at the age of 21. He presented a schizo-affective disorder with influence syndrome, auditory and visual hallucinations, labile mood with disinhibited and depressive periods. He was admitted in a psychiatry ward of a general hospital. An endocrinologist confirmed the diagnosis of KS and found very low blood testosterone levels. Besides lithium and risperidone which had already been introduced before the hospitalization, androgens (testosterone undecanoate) were very progressively given to Mr X with a daily psychiatric evaluation. One month after discharge, a major depressive episode led to the adjunction of citalopram. After one year of follow-up, Mr X shows increased social adjustment and enhanced interest; the influence syndrome has partially regressed and his mood is more stable. Discussion - In the years '60 and '70, systematic screenings in psychiatric hospitals have detected 1.3% KS among hospitalized boys, ie 10 times more than in the general population, and 0.6 to 1% KS among hospitalized men. A large variety of psychiatric disorders have been described. Boys presenting KS are usually described as shy, with little energy and initiative, and few friends. They cry more often than compares. Neuropsychological studies demonstrate significantly lower verbal IQ than controls, while performance IQ is generally normal and global IQ is in the normal range with large individual variations. Language acquisition is always delayed. However, agressiveness is not increased. In his follow-up study of 20 years, Nielsen at al found more psychiatric disorders among KS patients, compared to a group of hypogonadal patients at first examination (mean age=27 years). After 20 years follow-up, however, no significant difference remained between the two cohorts concerning the frequency of psychiatric hospitalizations or mental diseases. Several hypotheses have been proposed to explain psychological aspects of KS such as low levels of androgens during foetal and child development, personality disorder related to hypogonadism, delay of mitosis of cells with an extra X chromosome, but none of them is able to explain the specificity of psychological problems associated with KS. Concerning therapeutic aspects, specialists prone substitutive androgen therapy in case of too low testosterone blood levels, from the time of increase in FSH (around the age of 11-15). It prevents osteoporosis, backache and excessive tiredness often found in males with KS; testosterone also improves social drive, mood, concentration and ability at work. If KS diagnosis is made at adult age, androgen therapy has also shown some efficacy, though less than if started earlier. Due to the oral and written language problems of KS boys between 5 and 12 years of age, Graham et al. recommend anticipatory guidance for these boys. In addition, they insist on the importance of the information of the parents, language therapy, the reduction of the length of the instructions given by schoolmasters and specially stimulating and stable childhood conditions. Though it is generally thought that androgens increase agressiveness, we found no consistent data in litterature proving that the restoration of physiological androgen blood levels increases crimes nor aggressiveness. In the contrary, Miller and Sulkes described four cases of KS men presenting chronic fire-setting behaviors. Testosterone was introduced. For three of them, follow-up was available: their behavior seemed improved and none of them recurred. However, the initiation of androgen therapy for patients with severe psychiatric illness should be done very carefully. Conclusion - The Klinefelter's syndrome is frequent and, if not diagnosed (which seems to be the most common case), these men have higher risks to develop psychiatric disorders. Therefore, child psychiatrists and psychiatrists should evoke that diagnosis when they examine boys or men who present typical physical traits of KS (tallness, underdevelopped testes) associated to school problems and/or psychiatric disorders. Indeed, if the diagnosis is confirmed by an endocrinologist and a genetic testing, psychological follow-up and testosterone undecanoate treatment (in case of abnormal testosterone blood levels) should be initiated. This therapy generally improves physical well-being and improves mood, concentration, capacity at work. There is no consistent data in the litterature proving that restoring physiological testosterone blood levels would be dangerous for KS men presenting severe psychiatric troubles. However, this should be discussed in each situation with caution, and androgens should be introduced very progressively.

The mechanisms by which tumor cells extravasate to form metastasis remain controversial. Previous studies performed in vivo and in vitro demonstrate that the contact between tumor cells and the vascular wall impairs endothelium integrity. Here, we investigated the effect of breast adenocarcinoma MCF-7 cells on the apoptosis of human umbilical vein endothelial cells (HUVEC). TUNEL labeling, nuclear morphology, and DNA electrophoresis indicated that MCF-7 cells induced a two- to fourfold increase in HUVEC apoptosis. Caspase-3 activity was significantly enhanced. Neither normal cells tested (mammary epithelial cells, fibroblasts, leukocytes) nor transformed hematopoietic cells tested (HL60, Jurkat) induced HUVEC apoptosis. On the contrary, cells derived from solid tumors (breast adenocarcinoma, MDA-MB-231 and T47D; fibrosarcoma, HT 1080) had an effect similar to that of MCF-7 cells. The induction of apoptosis requires cell-to-cell contact, since it could not be reproduced by media conditioned by MCF-7 cells cultured alone or cocultured with HUVEC. Our results suggest that cells derived from solid tumors may alter the endothelium integrity by inducing endothelial cell apoptosis. On the contrary, normal or malignant leukocytes appear to extravasate by distinct mechanisms and do not damage the endothelium. Our data may lead to a better understanding of the steps involved in tumor cell extravasation.

Co-injection of fibroblasts with human epithelial breast-tumor MCF7 cells in the presence of Matrigel enhances tumor growth in nude mice. While most of the matrix metalloproteinases (MMPs) have been shown to be produced by stromal cells, tumor cells such as MCF7 cells are unable to produce MMPs. We therefore, hypothesized that the tumor-promoting effect of fibroblasts could be related to their production of MMPs. In order to inhibit stromal proteases, over-production of TIMP-2 was induced in MCF7 cells by in vitro retroviral-mediated gene transfer. TIMP-2-producing MCF7 cells were then co-injected with fibroblasts into nude mice. Alternatively, we evaluated the effect of Batimastat, a synthetic inhibitor of MMPs, on the tumorigenicity of MCF7 cells co-inoculated with fibroblasts into nude mice. Both physiological (TIMP-2) and synthetic (Batimastat) inhibitors of MMPs were able to abolish the tumor-promoting effect of fibroblasts. On the contrary, they failed to modulate the tumorigenicity of MCF7 cells injected alone. Interestingly, Matrigel from which low-molecular-weight proteins or growth factors had been removed failed to favor the tumorigenicity of MCF7 cells inoculated with fibroblasts. These findings emphasize the importance of fibroblasts in cancer progression, and suggest that their role could be related at least in part to production of proteases which can induce the release of factors from the extracellular matrix.

Stromelysin-3 (ST3; Basset, P., J.P. Bellocq, C. Wolf, I. Stoll, P. Hutin, J.M. Limacher, O.L. Podhajcer, M.P. Chenard, M.C. Rio, P. Chambon. 1990. Nature. 348:699-704) is a matrix metalloproteinase (MMP) expressed in mesenchymal cells located close to epithelial cells, during physiological and pathological tissue remodeling processes. In human carcinomas, high ST3 levels are associated with a poor clinical outcome, suggesting that ST3 plays a role during malignant processes. In this study we report the ST3 gene inactivation by homologous recombination. Although ST3 null mice (ST3-/-) were fertile and did not exhibit obvious alterations in appearance and behavior, the lack of ST3 altered malignant processes. Thus, the suppression of ST3 results in a decreased 7, 12-dimethylbenzanthracene-induced tumorigenesis in ST3-/- mice. Moreover, ST3-/- fibroblasts have lost the capacity to promote implantation of MCF7 human malignant epithelial cells in nude mice (P < 0.008). Finally, we show that this ST3 paracrine function requires extracellular matrix (ECM)-associated growth factors. Altogether, these findings give evidence that ST3 promotes, in a paracrine manner, homing of malignant epithelial cells, a key process for both primary tumors and metastases. Therefore, ST3 represents an appropriate target for specific MMP inhibitor(s) in future therapeutical approaches directed against the stromal compartment of human carcinomas.

Metalloproteinases and serine proteinases have been associated with tumor invasion and formation of metastasis which represent the major obstacles to cancer cure. The contribution of proteinases in these processes was initially thought to be the destruction of extracellular matrices. However, recent evidence suggests that they mainly affect tumor growth rather than invasion. Proteinases can indeed generate active matrix protein fragments, influence the release, the activation and the bioavailability of growth factors, and consequently modulate tumor cell growth, apoptosis and angiogenesis. Additionally, proteinases, their receptors and/or inhibitors can be directly involved in cell migration and in the processing or shedding of cell surface proteins. Further elucidation of the functions of proteinases is essential for the development of novel anticancer strategies.