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654 Vol. 1, 654–664, July 2003 Molecular Cancer Research Expression of Cyclin-Dependent Kinase 6, but not Cyclin-Dependent Kinase 4, Alters Morphology of Cultured Mouse Astrocytes Karen K. Ericson, David Krull, Peter Slomiany, and Martha J. Grossel Department of Biology, Connecticut College, New London, CT Abstract mechanism involving the accumulation of the p53 and p130 Disruption of the pRb pathway is a common mechanism growth-suppressing proteins (6), and activation of cdk6 in tumor formation. The D-cyclin-associated kinases, precedes cdk4 activation in T cells (7, 8). Also, differences in cyclin-dependent kinase (cdk) 4 and cdk6, are important subcellular localization of cdk4 and cdk6 and in the timing of regulators of the G1-S phase transition and are elevated nuclear localization have been noted in several cell types by in several types of cancers, including gliomas. To several researchers (9–12). Data from tumor studies also investigate potential functional differences in these suggest differences in these kinases. Cdk4, and not cdk6, is kinases, mouse astrocytes were taken from chimeric specifically targeted in melanoma (13, 14) while cdk6 activity mice and propagated in tissue culture. These multipolar has been found to be elevated in squamous cell carcinomas (15, tissue-culture astrocytes were infected with viruses 16) and neuroblastomas (17) without alteration of cdk4 activity. expressing either cdk4 or cdk6. Interestingly, expression Cumulatively, these data suggest that cdk4 and cdk6 have of cdk6 resulted in a distinct and rapid morphology unique functions that may be cell-type specific, temporally change from multipolar to bipolar. This change was not regulated, or developmentally distinct. observed in control astrocytes or in astroyctes infected The process of differentiation can be considered as coupled with cdk4. Several other differences in cdk4- and cdk6- processes including: (a) exit from the cell cycle and (b) infected cells were noted, including differential binding expression of tissue-specific genes (18). Differences in protein to a subset of cell-cycle inhibitor proteins and a distinct levels and activity of key cell cycle inhibitors have been noted pattern of subcellular localization of these kinases. on differentiation of some cells. For instance, the cdk inhibitor Immunoblot and immunofluorescence analyses revealed protein, p27kip1, has been identified as part of a timing that cdk6-infected astrocytes had an altered expression mechanism that causes oligodendrocyte precursor cells to profile of known markers of glial differentiation. withdraw from the cell cycle and undergo differentiation. In Together, these data indicate several important differ- this system, p27kip1 accumulation correlates with differentiation CIP1/WAF-1 ences between cdk4 and cdk6 that highlight unique of oliodendrocytes (19–21). Expression of p21 , functional roles for these cyclin-dependent kinases. another cyclin kinase inhibitor protein, has been associated with differentiation in a variety of tissues (22–26). In murine Introduction erythroleukemia (MEL) cells, inhibitors that blocked both cdk2 and cdk6 induced differentiation, but inhibition of cdk2 and The G1 cyclins and associated cyclin-dependent kinases are cdk4 did not (24). Thus, G1 phase inhibitors have been shown critical regulators of G1 phase of the cell cycle. Cell cycle progression is dependent on mitogenic signals that activate to be important in the process of differentiation in muscle, cyclin D and its associated kinases. Cyclin-dependent kinase blood, and adipocytes (18). (cdk) 4 and cdk6 partner with cyclin D proteins to We set out to study functional differences of cdk4 and phosphorylate the Retinoblastoma protein (pRb), allowing cdk6 using a previously described tissue-culture astrocyte onset of S-phase (for review, see Ref. 1). Since the initial model system. This system was chosen because these cells characterization of cdk6, the D-cyclin kinases have been widely contain a minimal number of genetic mutations and are thought to act redundantly to regulate the cell cycle through the physiologically relevant, since overexpression of cdk4 and phosphorylation of pRB (2–5). However, recent studies have cdk6 has been found in glioblastomas and astrocytomas (27– uncovered novel functions of cdk6 and several studies highlight 30). These primary astrocytes were isolated from newborn differences between cdk4 and cdk6. For instance, cdk6 plays a transgenic mice that had been engineered to express the Avian role in halting inappropriate cellular proliferation through a Leukosis Virus (ALV) receptor (TVA) downstream of the GFAP promoter (G-tva) (31). Only GFAP-producing cells expressed the viral receptor and thus only these cells could be infected with ALV (31, 32). Starting cultures of these Received 1/13/03; revised 4/22/03; accepted 5/7/03. astrocytes displayed a star-shaped astrocytic morphology with The costs of publication of this article were defrayed in part by the payment of multiple short projections from a central cell body. Impor- page charges. This article must therefore be hereby marked advertisement in tantly, these cultures did not resemble a bipolar, fibroblast-like accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: NSF under CAREER grant #9984454 to Martha J. Grossel. morphology typical of glial restricted precursors or type 1 Requests for reprints: Martha J. Grossel, Department of Biology, Box 5331, astrocytes [for further descriptions of glial differentiation, see Connecticut College, 270 Mohegan Ave., New London, CT 06320. Phone: (860) 439-5209; Fax: (860) 439-2519. E-mail: [email protected] Lee et al. (33)]. Because cdk4 expression has been well Copyright D 2003 American Association for Cancer Research. characterized in this system (32), these tissue culture Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. Molecular Cancer Research 655 astrocytes provided an ideal model in which to compare as a control. Astrocytes were infected with viral supernatants known functions, and to investigate novel functions of cdk4 from DF-1 cells producing ALV that expressed the cDNAs of and cdk6. Results presented here demonstrate that infection of AP, cdk4, or cdk6. Protein expression in transfected DF-1 cells cdk6, but not cdk4, is sufficient to cause morphogenesis of was confirmed by immunoblot and virus-containing super- cultured mouse astrocytes. natants from DF-1 cells were filtered onto G-tva mouse astrocytes and infected for 3 days. Protein expression of cdk4 and cdk6 in these astrocytes was confirmed by immunoblot Results (Fig. 1A). Endogenous cdk6 was not expressed to detectable Morphological Differences in Cdk4- and levels in these astrocytes. Cdk6-Infected Astrocytes Within days after infection, it was apparent that cdk6- To study overlapping and discreet functions of cdk4 and infected astrocytes had a strikingly different morphology than cdk6, G-tva mouse astrocytes were infected with either of the those infected with cdk4. The cdk6-infected cells displayed a two D-cyclin-associated kinases or alkaline phosphatase (AP) fibroblast-like, bipolar morphology. Digital images of these FIGURE 1. Morphology of AP-, cdk4-, and cdk6- infected astroyctes. A. Immunoblot of primary mouse astrocytes infected with AP, cdk4, or cdk6, as indicated. One hundred twenty micrograms of protein extract were loaded per lane. The blot is probed with a mixture of cdk4 and cdk6 antibodies to detect both proteins on one blot. B. Phase-contrast microscopy of infected astroyctes grown in monolayer. All images are taken at the same magnification. RCAS-cdk4/6 are RCAS- cdk4-infected astrocytes that were super-infected with RCAS-cdk6 virus. C, D. Scanning electron microscopy (SEM) of fixed astrocytes at lower (C) and higher (D) magnifications. Scale bars on images indicate size. In panel C, scale bar is 20 AM; in panel D, scale bar is 10 AM. Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. 656 Expression of Cdk6 Alters Morphology of Astrocytes cells grown in a monolayer are shown in Fig. 1B. Alkaline- Cdk4-Infected and Cdk6-Infected Cells Display Distinct phosphatase and cdk4-infected astrocytes displayed a star- Growth Curves shaped, flat morphology containing several short projections Changes in cell size associated with cdk6 expression might be emanating from a wide central cell body. In sharp contrast, a result of changes in cellular proliferation. To study cell division cdk6-infected mouse astrocytes displayed an elongated spindle- properties of cdk4 and cdk6, growth curves were generated. shaped morphology with bipolar processes. Cdk6-infected Infected astrocytes were grown until confluent and then were cells contain a narrow soma that is collapsed around the split, counted, and replated at a density of 1 Â 105 cells per plate nucleus and greatly extended projections emanating from and followed over a period of up to 120 days. Growth curves of the central cell body. In addition, many of the cells are phase- one of four independent infections are displayed in Fig. 2. The bright in the area surrounding the central cell body, a property growth curves of both cdk4- and cdk6-infected cells were not seen in AP and cdk4 cells. The projections that extended remarkably consistent across all infections; in four separate trials from the cdk6 cells formed connections between adjacent cells of cdk4 and cdk6 infections, these cells escaped

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