Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1.
The major regulator of the gap-1/synthesis phase (G(1)/S) cell cycle checkpoint is the retinoblastoma protein (pRb), and this is regulated in part by the activities of cyclin-dependent kinase (cdk)-4 and the D cyclins. Surprisingly, given the potential importance of beta-cell replication for islet replacement therapy, pRb presence, phosphorylation status, and function have not been explored in beta-cells. Here, adenoviruses expressing cdk-4 and cyclin D(1) were used to explore rat and human pRb phosphorylation and beta-cell cycle control. pRb is present in rat and human islets, and overexpression of cyclin D(1)/cdk-4 led to strikingly enhanced pRb phosphorylation in both species. Combined overexpression of both cdk-4 and cyclin D(1) caused a threefold increase in [(3)H]thymidine incorporation. This increase in proliferation was confirmed independently using insulin and bromodeoxyuridine immunohistochemistry, where human beta-cell replication rates were increased 10-fold. Cdk-4 or cyclin D(1) overexpression did not adversely effect beta-cell differentiation or function. The key cell cycle regulatory protein, pRb, can be harnessed to advantage using cyclin D(1)/cdk-4 for the induction of human and rodent beta-cell replication, enhancing replication without adversely affecting function or differentiation. This approach will allow detailed molecular study of the cellular mechanisms regulating the cell cycle in beta-cells, beta-cell lines, and stem cell-derived beta-cells.
Cozar-Castellano
Irene
I
Division of Endocrinology and Metabolism, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA. cozari@msx.dept-med.pitt.edu
Takane
Karen K
KK
Bottino
Rita
R
Balamurugan
A N
AN
Stewart
Andrew F
AF
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000235:genetics / Q000378:metabolism*; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism*; D007700:Kinetics; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; D019938:Cyclin D1; C495900:CDK4 protein, human; C495903:Cdk4 protein, rat; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases