Study of events leading cellular senescence to human mammary epithelial cancer cells in vitro.

ABSTRACT

Role of various growth regulatory factors in inducing senescence in cultured HMEC cells have been investigated in ten cases of breast cancer. The histological grade of tumour cells is found to play significant role in controlling the proliferation and phenotypic charateristics of cultured HMEC cells during primary culture and also number of subsequent passages resulting in complete cellular senescence in them. Effects of conditioned media (CM) collected from primary and senescent cultures of these HMEC cells had also been studied on proliferation of their own HMEC cells used as target cells, to evaluate the role of various autocrine growth factors produced by them. Significant increase in proliferation of target cells was noticed on their exposure to CM from senescent cultures, while cessation of their proliferation was found on their exposure to CM from senscent cultures, suggesting that HMEC cells produce growth promoting factors during primary culture and growth inhibitory factors on subsequent passages, responsible for inducing features of cellular senescence in them. The role of epidermal growth factors (EGF) and transforming growth factors (TGF) alpha and beta as autocrine factors in inducing senescence of cultured HMEC cells were also investigated. Deletion of EGF from growth media initially caused decreased proliferation to target HMEC cells, followed by improvement in their proliferation. Supplementation of growth media by TGF-alpha induced significant increase in proliferation of target cells. Addition of epidermal growth factors receptor (EGFR) antibody to cells exposed to media devoid of EGF and media supplemented with TGF-alpha showed marked suppression of proliferation of target cells. The morphologic and phenotypic characteristics of target HMEC cells exposed to TGF-alpha were also found similar to those HMEC cells grown during primary culture, suggesting autocrine production of EGF and TGF-alpha by cultured HMEC cells during primary culture. Supplementation of TGF-beta to growth media induced marked suppression of proliferation to target cells along with morphologic and phenotypic features of terminal differntiation or senescence. Exposure of senescent cells to media supplemented with EGF and TGF-alpha could not induce their proliferation. This suggest that HMEC cells on subsequent passages undergo some genetic and phenotypic alterations resulting in production of growth inhibitory factor like TGF-beta which induces cessation of their proliferation alone with features of senescence.