Search

In addition to its mitogenic and survival actions, recent evidence indicates that IGF-I can enhance DNA repair, implying IGF activity may limit the efficacy of many therapeutic strategies that rely on induction of DNA damage. Although the individual pathways by which DNA damage and IGF-I act are well understood, the cross-talk between these signaling events is not well defined. We examined the effects of DNA damage on the IGF-I response of MCF-7 breast cancer cells. Cells were exposed to the UV mimetic, 4-NQO, or the gamma-irradiation mimetic and chemotherapeutic drug, bleomycin; cellular proliferation was assessed by cell counting, tritiated thymidine incorporation and FACS cell cycle analysis. Although IGF-I acutely suppressed the p53 response to both agents, it subsequently enhanced the chronic increase in p53 and p21(WAF1/Cip1), resulting in cell cycle arrest; however, no apoptosis was observed. Use of specific inhibitors demonstrated that PI3 kinase was utilized with p38 MAPK to induce the p53 response to DNA damage, but was also utilized by IGF-I to diminish the acute p53 response. In addition, p21WAF1/Cip1 was increased by IGF-I, which has previously been shown to contribute to the mitogenic response. Here we demonstrate that with DNA damage IGF-I can also enhance the chronic p53-dependent increase in p21WAF1/Cip1, culminating in growth arrest. Overall, we have shown that PI3 kinase and p21WAF1/Cip1 play dual roles in mediating the mitogenic response to IGF-I, but these are both switched by cellular DNA damage to mediate a growth arrest., ((c) 2005 Wiley-Liss, Inc.)

Variability in response to chemotherapy is poorly understood. Paclitaxel-induced apoptosis was assessed in human Hs578T breast cancer cells, using the MTT assay, cell counting, morphological features and flow cytometry. Pre-dosing cells with non-glycosylated insulin-like growth factor binding protein-3 (ngIGFBP-3) had no effect on the cells per se but accentuated paclitaxel-induced apoptosis. The apoptotic pathway was further examined by measuring caspase-3 activity in cell lysates at time points over 48 hr after dosing with paclitaxel. Activity increased significantly, and Western immunoblots for caspase-3 in conditioned media showed that the inactive precursor decreased after incubation with paclitaxel. Endogenous production of IGFBP-3 by the cells after incubation with paclitaxel was evaluated using Western ligand blotting, specific IGFBP-3 immunoblotting and radioimmunoassay. Paclitaxel increased endogenous IGFBP-3, which was further increased if the cells had been pre-dosed with ngIGFBP-3. These findings suggest that IGFBP-3 may be an important modulator of paclitaxel-induced apoptosis.

Neoplastic transformation is characterised by an imbalance in favour of cell growth over programmed cell death (apoptosis). The tumour-suppressor gene p53, responsible for maintaining cell-cycle control, is mutated in the majority of human cancers. Loss of function of the target genes of p53 are therefore important in tumourigenesis. One such target gene is the insulin-like growth factor binding protein-3 (IGFBP-3), an extracellular protein responsible for the carriage of IGF-I but which can act independently of IGF-I, inhibiting cell growth and enhancing apoptosis. Using the KYSE 190 oesophageal carcinoma cell line, we have demonstrated that IGFBP-3 alone has no effect on cell growth or cell survival. However, it significantly enhanced apoptosis, with a 67% increase in the pre-G1 peak on flow cytometry following UV irradiation. The increase in p53 was enhanced and prolonged when cells are stressed in the presence of IGFBP-3. These data suggest an autocrine/paracrine feedback loop exists between IGFBP-3 and p53, which may provide the social control necessary to maintain normal tissue homeostasis.

Despite the increase in laser therapy, concern remains that sublethal treatment of pre-malignant lesions may adversely affect the biological behaviour of surviving cells. Integrin receptors mediate interaction of cells with the extracellular matrix and their occupation leads to focal adhesion kinase (FAK) activation. Using our previously established model we have now investigated subcellular changes and compared integrin and FAK concentrations, the degree of FAK phosphorylation and its association with the beta1 integrin in laser vs. non-laser treated cells. We treated cells with laser generated from a frequency doubled Q-switched (Nd:YAG) laser system (532 nm) at 0.4 J/cm2 twice per week for 4 weeks. Using cell lysates we performed Western immunoblotting 24 hr later to detect integrin subunits and FAK proteins and immunoprecipitation to investigate FAK phosphorylation and its association with beta1. Cell morphology was examined using electron microscopy. SK23 and G361 cells exhibited an 3.4- and 11.2-fold increase, respectively, in FAK protein following laser treatment. FAK phosphorylation in SK23 cells was increased by 82%, whereas FAK phosphorylation in G361 cells was reduced slightly (2%). Furthermore, both alpha3 and 4 integrins were up-regulated, by approximately 4-fold and 7- to 9-fold, respectively. In addition, the beta1 integrin was proteolysed in both cell lines and the levels of FAK associated with beta1 was increased (2.1- and 2.7-fold, respectively). Finally, laser treatment of SK23 cells caused an increased number of cell processes. Sublethal 532 nm laser light thus induces changes in integrin and FAK concentrations and subsequently influences cellular attachment and morphology.