Synchronization in G0/G1 enhances the mitogenic response of cells overexpressing the human insulin receptor A isoform to insulin

Evaluating mitogenic signaling specifically through the human insulin receptor (IR) is relevant for the preclinical safety assessment of developmental insulin analogs. It is known that overexpression of IR sensitizes cells to the mitogenic effects of insulin, but it is essentially unknown how mitogenic responses can be optimized to allow practical use of such recombinant cell lines for preclinical safety testing. We constitutively overexpressed the short isoform of the human insulin receptor (hIR-A, exon 11-negative) in L6 rat skeletal myoblasts. Because the mitogenic effect of growth factors such as insulin is expected to act in G0/G1, promoting S-phase entry, we developed a combined topoinhibition + serum deprivation strategy to explore the effect of G0/G1 synchronization as an independent parameter in the context of serum deprivation, the latter being routinely used to reduce background in mitogenicity assays. G0/G1 synchronization significantly improved the mitogenic responses of L6-hIR cells to insulin, measured by 3H-thymidine incorporation. Comparison with the parental L6 cells using phospho-mitogen-activated protein kinase, phospho-AKT, as well as 3H-thymidine incorporation end points supported that the majority of the mitogenic effect of insulin in L6-hIR cells was mediated by the overexpressed hIR-A. Using the optimized L6-hIR assay, we found that the X-10 insulin analog was more mitogenic than native human insulin, supporting that X-10 exhibits increased mitogenic signaling through the hIR-A. In summary, this study provides the first demonstration that serum deprivation may not be sufficient, and G0/G1 synchronization may be required to obtain optimal responsiveness of hIR-overexpressing cell lines for preclinical safety testing

Insulin-like growth factors (IGF) I and II utilize different calcium signaling pathways in a primary human parathyroid cell culture model

Background In most cell types, influx of calcium (Ca2+) induces a growth or secretory response. The opposite occurs in parathyroid (PTH), cells where there is an inverse relationship between intracellular Ca2+ concentration and PTH secretion. We have examined the effects of calcium channel and metabolism modulators on insulin-like growth factors (IGFs) in a parathyroid cell culture model. Methods Cell cultures were prepared from 9 patients undergoing operation for hyperparathyroidism. Following adhesion, the cells were transferred to serum-free medium and dosed with IGF I, II ± ethyleneglycol-bis(β-aminoethyl)-N, N, N′,N′-tetraacetic acid (EGTA), nifedipine, nickel, 2-aminoethoxy-diphenylborate (2-APB), or dantrolene. Proliferation (96 hours) was assessed by measuring tritiated thymidine incorporation and PTH release (1 and 3 hours) assayed by IRMA. Results Both IGF I and II increased DNA synthesis to 162.8% ± 10.6% (SEM) and 131.1% ± 7.7%, respectively (P < 0.05). EGTA at 0.2 mmol (ionized Ca2+ 0.2mmol) did not affect the response to both IGFs. EGTA at 2 mmol (ionized Ca2+ 0 mmol) reduced the DNA synthesis of IGF I and II to 29% and 26%, respectively (P < 0.05). Nifedipine and nickel (nonspecific Ca2+ channel blocker) were equally potent in negating the mitogenic effects of both IGFs. 2-APB (IP3R blocker) reduced the basal DNA synthesis to 51.3% ± 8.4% but had no effect on either IGF. Dantrolene (ryanodine receptor blocker) negated IGF II induced mitogenisis (74.2% ± 6.7%) and partially inhibited IGF I mitogenesis (123% ± 6%) (P < 0.05). The rate of PTH secretion was greater after IGF II stimulation than after IGF I stimulation. Conclusions IGFs I and II induce mitogenesis by different calcium signaling pathways. These data suggest that parathyroid cells may utilize different calcium signaling pathways to distinguish growth factors and serum calcium changes

Computational Modelling of Cancer Development and Growth:Modelling at Multiple Scales and Multiscale Modelling

MAJC and CKM gratefully acknowledge support of EPSRC grant no. EP/N014642/1 (EPSRC Centre for Multiscale Soft Tissue Mechanics – With Application to Heart & Cancer).In this paper, we present two mathematical models related to different aspects and scales of cancer growth. The first model is a stochastic spatiotemporal model of both a synthetic gene regulatory network (the example of a three-gene repressilator is given) and an actual gene regulatory network, the NF- κB pathway. The second model is a force-based individual-based model of the development of a solid avascular tumour with specific application to tumour cords, i.e. a mass of cancer cells growing around a central blood vessel. In each case, we compare our computational simulation results with experimental data. In the final discussion section, we outline how to take the work forward through the development of a multiscale model focussed at the cell level. This would incorporate key intracellular signalling pathways associated with cancer within each cell (e.g. p53–Mdm2, NF- κB) and through the use of high-performance computing be capable of simulating up to 109 cells, i.e. the tissue scale. In this way, mathematical models at multiple scales would be combined to formulate a multiscale computational model.PostprintPeer reviewe