Cytoplasmic-nuclear trafficking of G1/S cell cycle molecules and adult human ß-cell replication: a revised model of human ß-cell G1/S control.

Harnessing control of human ß-cell proliferation has proven frustratingly difficult. Most G1/S control molecules, generally presumed to be nuclear proteins in the human ß-cell, are in fact constrained to the cytoplasm. Here, we asked whether G1/S molecules might traffic into and out of the cytoplasmic compartment in association with activation of cell cycle progression. Cdk6 and cyclin D3 were used to drive human ß-cell proliferation and promptly translocated into the nucleus in association with proliferation. In contrast, the cell cycle inhibitors p15, p18, and p19 did not alter their location, remaining cytoplasmic. Conversely, p16, p21, and p27 increased their nuclear frequency. In contrast once again, p57 decreased its nuclear frequency. Whereas proliferating ß-cells contained nuclear cyclin D3 and cdk6, proliferation generally did not occur in ß-cells that contained nuclear cell cycle inhibitors, except p21. Dynamic cytoplasmic-nuclear trafficking of cdk6 was confirmed using green fluorescent protein-tagged cdk6 and live cell imaging. Thus, we provide novel working models describing the control of cell cycle progression in the human ß-cell. In addition to known obstacles to ß-cell proliferation, cytoplasmic-to-nuclear trafficking of G1/S molecules may represent an obstacle as well as a therapeutic opportunity for human ß-cell expansion.

cMyc is a principal upstream driver of beta-cell proliferation in rat insulinoma cell lines and is an effective mediator of human beta-cell replication.

Adult human ß-cells replicate slowly. Also, despite the abundance of rodent ß-cell lines, there are no human ß-cell lines for diabetes research or therapy. Prior studies in four commonly studied rodent ß-cell lines revealed that all four lines displayed an unusual, but strongly reproducible, cell cycle signature: an increase in seven G(1)/S molecules, i.e. cyclins A, D3, and E, and cdk1, -2, -4, and -6. Here, we explore the upstream mechanism(s) that drive these cell cycle changes. Using biochemical, pharmacological and molecular approaches, we surveyed potential upstream mitogenic signaling pathways in Ins 1 and RIN cells. We used both underexpression and overexpression to assess effects on rat and human ß-cell proliferation, survival and cell cycle control. Our results indicate that cMyc is: 1) uniquely up-regulated among other candidates; 2) principally responsible for the increase in the seven G(1)/S molecules; and, 3) largely responsible for proliferation in rat ß-cell lines. Importantly, cMyc expression in ß-cell lines, although some 5- to 7-fold higher than normal rat ß-cells, is far below the levels (75- to 150-fold) previously associated with ß-cell death and dedifferentiation. Notably, modest overexpression of cMyc is able to drive proliferation without cell death in normal rat and human ß-cells. We conclude that cMyc is an important driver of replication in the two most commonly employed rat ß-cell lines. These studies reverse the current paradigm in which cMyc overexpression is inevitably associated with ß-cell death and dedifferentiation. The cMyc pathway provides potential approaches, targets, and tools for driving and sustaining human ß-cell replication.