Therapeutic Differentiation of Tumor-derived Insulin-producing Cells Selected for Resistance to Diabetogenic Drugs

Differentiation therapy has been proposed as a new approach to selectively engage the process of tumor cell differentiation during chemotherapy of cancer. Our recent in vitro study suggests that such an approach can be extended and utilized for the selection of tumor-derived insulin-producing cells for transplantation. Repeated treatment with streptozotocin selected toxin resistant subpopulation of insulin producing tumor RINmS cells, characterized increased level of insulin content and secretion. In the present study RINmS cells were found to have higher glucose sensitivity and insulin response compared with parental RINm cells. In addition, compounds known to induce elevated level of cAMP beta-cells, such as isobutyl methyl xanthine, and forskolin, potentiated glucose-induced insulin secretion of RINmS, but had no effect on the naive parental RINm cells. These experiments suggest that differentiation therapy can be utilized for engineering insulin producing cells with improved defense and secretory mechanisms.


INTRODUCTION
Tumor derived insulin-producing beta-cell lines constitute a potential source for islet transplantation. E1'21 To achieve long-term functioning, transplanted beta cells must posses a high level of differentiation to allow adequate glucose sensing, insulin processing and secretion, and an effective defense mechanism.
Recently, differentiation therapy has been proposed as a new approach to selectively engage the process of tumor cell differentiation during chemotherapy of cancer. E3J According to this approach, cytotoxic agents, including chemical compounds and cytokines used in cancer therapy, can induce drug resistance, but in certain conditions, can also lead to elimination of tumorigenic cells and to recovery of normal cell homeostasis. [4][5][6] We have recently found that repeated, in vitro treatment, with streptozotocin (STZ), an antineoplastic compound utilized for the treatment of *Corresponding author. Fax: 972-3-9211478, Tel: 972-3-9376280. e-mail: pvardi@post.tau.ac.il patients with insulinoma, selects toxin resistant subpopulation of insulin-producing RINmS tumor cells. [7,s] The higher resistance to both STZ and alloxan (AL), was found to be at least partially dependent on the lower expression of the glucose transporter 2 (GLUT-2) in the selected cells. Despite the lower expression of GLUT-2 in the resistant cells, their functional capacity, reflected by the increased intracellular insulin content and insulin release, did not seem to be impaired. In the present study we further evaluated the insulin response to different secretagogues, and used the proliferation capacity of RINmS cells as a marker of differentiation stage.

Cell Culture and Selection Procedure
The RINm cells, which have been previously described in detail, [9] were cultured in RPMI 1640 medium, supplemented with 10% FCS, 2mmol/1 L-glutamine, 100 IU penicillin and 100tg/ml streptomycin. Cells were grown in plastic tissue culture flasks at 37C in 95% humidified air with 5% CO2. The toxin resistant cells (RINmS) were developed by exposing cells to 10 mM of STZ in 2 passages, as previously described. [

RESULTS AND DISCUSSION
We previously demonstrated that repeated exposure of tumor-derived insulin-producing RINm cells to high doses of STZ leads to selection of a cell subpopulation (RINmS) with a higher resistance not only to STZ, but also to AL. I7,s] The higher cell defense property of the surviving cells was found to be associated with a decreased expression of GLUT-2, which acts as a transporter of STZ and AL. In addition to their modified defense potential, resistant RINmS cells were found also to contain and release significantly higher level of insulin when compared with their untreated parental RINm cells. The finding of low GLUT-2 expression in the highly resistant and more differentiated RINmS cells is unexpected and opposes that of high level of GLUT-2 and extreme cell sensitivity to diabetogenic toxins reported in experiments with rodent islets. Such controversy could result from the different cell pathways of natural selection in the whole animals, and the artificially-induced in vitro selection of RINmS cells.
We further evaluated the cell functional capacity and found that RINmS cells are more sensitive to increasing concentrations of glucose when compared with RINm cells. Thus, incubation with 5mM of glucose alone induced a twofold increase in insulin secretion in RINmS and had no effect in RINm cells (Fig. 1). Furthermore, IBMX and FSK, compounds which induce elevation of cAMP level in beta-cells, lead to a 4-fold increase of glucose (5 mM) induced insulin secretion in the toxin-resistant RINmS but not in the parental cells (Fig. 2). These data suggest that the lower GLUT-2 expression of RINmS cells, previously reported, IS] is probably sufficient to maintain the mechanism of glucose induced insulin secretion. It is possible that the higher cellular insulin content of RINmS cells, its improved regulation and secretion is probably due to transition of this cell population to a higher cell differentiation stage. This possibility is further supported by our finding of lower cell proliferative activity in RINmS when compared with parental RINm cells (Fig. 3). A similar induction of drug resistance in association with cell differentiation and growth suppression was previously reported in selected tumor cells following antineoplastic drug treatment. I3,5 Over-expression of multi drug resistance genes (mdrl), and decreased expression of nuclear transcription factor genes (c-myc), which control tumorigenic development are believed to be the underlying molecular mechanisms which activate such cellular pathway. Ilol We speculate that the alkylating agent STZ, not only induces resistance to diabetogenic drugs, but also induces increased DNA methylation, which in turn reinforces differentiation process in selected cells.