A novel messenger ribonucleic acid homologous to human MAGE-D is strongly expressed in rat Sertoli cells and weakly in Leydig cells and is regulated by follitropin, lutropin, and prolactin.

We have cloned a novel complementary DNA whose expression was decreased in rat Sertoli cell cultures after treatment with FSH. This complementary DNA encodes a protein of 570 amino acids and shares 92% homology with the human MAGE-D protein. In contrast to other MAGE genes (A, B, or C), we have shown that MAGE-D expression was ubiquitous in healthy rat tissues. In the seminiferous tubules, the MAGE-D was expressed in Sertoli cells but not in germ cells as demonstrated by RT-PCR and in situ hybridization, whereas for the other MAGE genes, expression has been shown to be restricted to germ cells. Interestingly, MAGE-D was also detected for the first time in the female gonad by Northern blotting. In MLTC-1 cells (mouse Leydig tumor cell line-1), LH and PRL stimulated MAGE-D expression. Using hypophysectomized rats, it was confirmed that FSH decreased MAGE-D expression, whereas LH and PRL increased MAGE-D messenger RNA level in the whole testis most probably through a direct action on Leydig cells. As MAGE-D is present in both the seminiferous compartment and interstitium and hormonally regulated in each, it is possible that it has specific functions in each compartment during the development and the maintenance of the testis.

TNP-470, a potent angiogenesis inhibitor, amplifies human T lymphocyte activation through an induction of nuclear factor-kappaB, nuclear factor-AT, and activation protein-1 transcription factors.

TNP-470, an angiogenesis inhibitor derived from fumagillin, is foreseen as a promising anti-cancer drug. Its effectiveness to restrain tumor growth and its lack of major side effects have been demonstrated in several animal models and have led the drug to reach phase III clinical trials. Beside its antiangiogenesis activities, TNP-470 exhibits several effects on the immune system. We had shown previously that TNP-470 stimulated B lymphocyte proliferation through an action on T cells. In this study, we examined the cellular and molecular modifications induced by TNP-470 in normal human T lymphocytes. Transmission electron microscopic examination of PHA/TNP-470-treated T cells revealed significant morphologic modifications when compared with PHA-treated control T cells. TNP-470 induced indeed an important and significant increase of the nuclear size as well as major nuclear chromatin decondensation. This observation indicated that TNP-470 amplified T-cell activation and led us to investigate its effects on the activation of transcription factors involved in T-cell activation. Using electrophoretic mobility shift assays, we have demonstrated that TNP-470 amplifies and extends the DNA-binding activity of nuclear factor-AT, nuclear factor-KB, and activation protein-1 in T cells. Furthermore, the angioinhibin significantly increased the secretion of IL-2 and IL-4. Our data demonstrate that TNP-470 amplifies the activation of T cells. This effect, whose molecular mechanisms remain to be elucidated, has to be taken into account in the assessment of the antitumor effect of the drug.

A novel gene overexpressed in the prostate of castrated rats: hormonal regulation, relationship to apoptosis and to acquired prostatic cell androgen independence.

We have identified a novel complementary DNA (cDNA) corresponding to a gene overexpressed in the rat ventral prostate after castration. This cDNA displays 89.4% identity with 453 bp of a mouse EST and 81.5% identity with 157 bp of a human EST and was named PARM-1 for prostatic androgen-repressed message-1. The complete cDNA is 1187 bp long and codes for a protein of 298 amino acids that contains four potential glycosylation sites and three half cystinyl residues. The PARM-1 gene was found to be expressed at quite low levels in most rat tissues including those of the urogenital tract. The kinetic of induction of PARM-1 gene in the prostate was highly correlated to the development of apoptosis in the whole organ. Supplementation of castrated animals with androgens reversed both the process of apoptosis and the overexpression of PARM-1 gene. Supplementation with estrogens did not result in an increase in the PARM-1 messenger RNA levels when compared with the castration alone. However, the treatment resulted in a more rapid return to intact levels in the castrated plus estrogen group. When apoptosis of testis and prostate was induced in vivo by hypophysectomy, it was found that PARM-1 was only overexpressed in the prostate. Therefore, PARM-1 seems to be regulated by androgens only in the prostate. Using in situ hybridization and immunohistological techniques, we have shown that PARM-1 gene product is found exclusively in the epithelial cells of involuting prostate. Analysis by flow cytometry of MAT LyLu epithelial cells transiently expressing PARM-1 protein did not allow us to demonstrate a direct effect of PARM-1 gene overexpression on the programmed death of the transfected cells. Treatment of MAT LyLu cells by transforming growth factor-beta induced apoptosis but had no effect on PARM-1 production. However PARM-1 protein has been detected by Western blotting in various cell lines such as MAT LyLu, MAT Lu, and PIF, which are androgen independent. This would suggest that PARM-1 gene product would be a marker for acquired androgen-independence of these tumor cells.

Expression and modulation of homeobox genes from cluster B in endothelial cells.

Angiogenesis is a complex phenomenon likely to be under the strict control of a group of transcription factor(s). Homeobox (HOX)-containing proteins have been identified as regulators controlling the coordinated expression of genes involved in organ development and tissue differentiation. In this study, we have demonstrated that human umbilical vein endothelial cells (HUVEC) express 8 of the 10 HOX genes contained in cluster B. Treatment of HUVEC with tissue plasminogen activator (TPA), an agent known to induce morphologic changes in endothelial cells, or vascular endothelium growth factor (VEGF), a proliferative and angiogenesis inducer, results in a specific time-dependent modulation of the eight HOX genes identified. Interestingly, neither basic fibroblast growth factor, an endothelial proliferative agent, nor TNP-470, a fumagillin derivative with potent antiendothelial cell proliferation properties, affected expression of these HOX genes. Specific modulation of HOX genes by differentiating agents but not by proliferative or antiproliferative molecules suggests that they could be involved in the control of the genetic program that coordinates the construction of new blood vessels.

In vitro and in vivo stimulation of the murine immune system by AGM-1470, a potent angiogenesis inhibitor.

AGM-1470, a potent angiogenesis inhibitor, is already engaged in phase I clinical trials because of its effectiveness to restrain tumor growth and its lack of major side effects. Recently, we showed that AGM-1470 stimulates in vitro human B lymphocyte proliferation through T lymphocytes. These data prompted us to explore the in vivo effects of AGM-1470 on the immune system in a mouse model. In this study, we showed that AGM-1470, in synergy with phytohemagglutinin, stimulates the proliferation of murine lymphocytes isolated from lymph nodes. This effect was similar to the one observed with human lymphocytes. When injected subcutaneously or intraperitoneally into mice at pharmacological doses, AGM-1470 induced a significant increase of axillary and mesenteric lymph nodes, respectively. Histological and morphological analyses showed that this phenomenon is mostly due to a hyperplasia of the germinal centers. On average, the area of the germinal center of lymph nodes from AGM-1470-treated mice were three times larger than in lymph nodes from control mice. Interestingly, no effect was observed when AGM-1470 was injected subcutaneously into T-deficient nude mice. Our data demonstrate that AGM-1470 stimulates B cell proliferation in vivo as suggested by the in vitro experiments. This effect should be taken into account in the follow-up of patients treated with this molecule and calls for additional studies to determine the biological consequences of such a stimulation on the host immune system.

The potent angioinhibin AGM-1470 stimulates normal but not human tumoral lymphocytes.

BACKGROUND: AGM-1470 is a newly synthesized molecule developed as an analog of the potent anti-angiogenic fumagillin. Its efficacy in restraining tumor growth in vivo and the absence of major side effects have already led to phase I clinical trials in patients with solid cancers. However, neither the exact mechanisms of action of AGM-1470 nor its effects on the host of normal cells have been extensively studied. Recently, we showed that AGM-1470 enhanced the proliferation of B lymphocytes in the presence of T cells. Since AGM-1470 could potentially be used in patients with lymphoma, it was urgent to test the effect of the molecule on the proliferation of tumor lymphocytes. METHODS: The possible effect of AGM-1470 on the proliferation of normal or tumor lymphocytes was evaluated by thymidine-incorporated assays. Normal T and B lymphocytes were purified from human tonsils. The tumor lymphocytes used in the study were Molt 3, Molt 4 and Jurkatt cell lines for the T lineage and Daudi and Radji cell lines for the B lineage. RESULTS: As shown previously, AGM-1470 stimulates the proliferation of normal B lymphocytes through an action on normal T cells. THe angioinhibin was ineffective ont eh proliferation of both T and B transformed cells. Moreover, in the presence of the drug, tumor T cells co-cultured with normal B lymphocytes did not induce any increase in B cell proliferation, as previously observed with normal T lymphocytes. Inversely, tumor B cells co-cultured with normal T lymphocytes were insensitive to the drug. CONCLUSIONS: Our results demonstrate that AGM-1470 is ineffective on lymphoid tumor cell proliferation and could potentially be safely administered to lymphoma patients.