Recent evidence indicates that growth hormone-releasing hormone (GHRH) functions as an autocrine/paracrine growth factor for various human cancers. A splice variant (SV) of the full-length receptor for GHRH (GHRHR) is widely expressed in various primary human cancers and established cancer cell lines and appears to mediate the proliferative effects of GHRH. To investigate in greater detail the role of SV1 in tumorigenesis, we have expressed the full-length GHRHR and its SV1 in MCF-7 human breast cancer cells that do not possess either GHRHR or SV1. In accordance with previous findings, the expression of both GHRHR and SV1 restored the sensitivity to GHRH-induced stimulation of cell proliferation, with SV1 being more potent than the GHRHR. Furthermore, MCF-7 cells transfected with SV1 proliferated more quickly than the controls, even in the absence of exogenously added GHRH, suggesting the existence of intrinsic, ligand-independent activity of SV1 after its transfection. In agreement with the stimulation of cell proliferation, the levels of proliferation markers cyclin D1, cyclin E, and proliferating cell nuclear antigen were elevated in MCF-7 cells treated with GHRH, cultured in both serum-free and serumcontaining media. In addition, SV1 caused a considerable stimulation of the ability of MCF-7 cells to grow in semisolid medium, an assay considered diagnostic for cell transformation. Collectively, our findings show that the expression of SV1 confers oncogenic activity and provide further evidence that GHRH operates as a growth factor in breast cancer and probably other cancers as well.carcinogenesis ͉ intrinsic activity ͉ transfection G rowth hormone-releasing hormone (GHRH), produced by the hypothalamus, regulates the secretion of growth hormone (GH) by binding to specific receptors in the pituitary (1). In addition to this neuroendocrine action, recent studies also indicate that GHRH functions as an autocrine/paracrine growth factor in various cancers (2-5). The evidence for this concept includes the detection of GHRH expression in several extrapituitary tissues, especially primary human cancers and experimental tumors, and the ability of antagonistic analogs of GHRH to inhibit cancer cell growth by mechanisms that do not involve the GH-induced stimulation of production of hepatic insulin-like growth factor I (2-6).Our understanding of how GHRH acts in extrapituitary tissues, and especially cancers, is restricted by poor functional characterization of the receptor(s) that mediates its mitogenic effects, especially in view of the limited expression of GHRH receptor (GHRHR) by extrapituitary tissues (7-9). Recently, it has been demonstrated that a splice variant (SV) of GHRHR (designated SV1, which differs from GHRHR in a short segment of the extracellular ligand-binding domain of the receptor protein) is widely expressed by different primary human and experimental cancers and can elicit mitogenic responses in the presence of GHRH (10-12). The suppression of the expression of SV1 by antisense RNA-based approache...

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p16 (INK4a) is a known negative regulator of the cell cycle acting up-stream of Rb to inhibit cellular growth. While the contribution of p16 to the tumorigenic process has been extensively studied, little is known about its role in the cellular differentiation process of normal cells. p16 expression in mammary gland epithelial cells and its possible mediation by DNA methylation was explored. The mammary glands from female mice (mus musculus) at distinct developmental stages (virgin, day 10 of lactation and day 3 of involution) were used. The expression pattern of p16 and the DNA methylases, DNMT1, 3a and 3b was investigated by semi-quantitative RT-PCR, in situ hybridization (ISH) and immunohistochemistry. The p16 methylation status was assesed by methylation-specific PCR (MSP). p16 was differentially expressed during distinct developmental stages and its transcriptional regulation was DNA methylation-independent, which was also corroborated by the expression pattern of the three known DNA methyltransferases (DNA MTase). The p16 expression level was elevated during involution compared to the corresponding expression level during lactation. p16 is differentially expressed during normal mammary gland development, which is not mediated by DNA methylation.

While the presence of stromal fibroblasts within malignant tumors was established many decades ago, it is only recently that their role in tumorigenesis has begun to unravel. Probably the most important finding in this line of research is that, contrary to the conventional notion that views them as a static entity, stromal fibroblasts progress in parallel with the cancer cells, interacting continuously with them and affecting the kinetic profile and the morphology of the tumor. However, while the tumor is highly dependent on the stromal fibroblast during the early stages of disease development, it is likely that it abolishes this dependency at later stages. The aim of this article is to outline the evidence implying this differential dependency of the cancer cells against the stromal fibroblasts, and to discuss their implication in the management of the disease, especially in view of the evolving concept of stroma-targeting anticancer therapies.