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Molecular Cancer Research 2:105-114 (2004)
© 2004 American Association for Cancer Research

Cell Cycle, Cell Death, and Senescence

Cyclin-Dependent Kinase 6 Inhibits Proliferation of Human Mammary Epithelial Cells1

Joseph J. Lucas, Joanne Domenico and Erwin W. Gelfand

Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO

Requests for reprints: Joseph J. Lucas, Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. Phone: (303) 398-1206; Fax: (303) 270-2182. E-mail: lucasj{at}njc.org


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Many defects in cancer cells are in molecules regulating G1-phase cyclin-dependent kinases (cdks), which are responsible for modulating the activities of Rb family growth-suppressing proteins. Models for understanding how such defects affect proliferation assume that cdks are responsible for sequentially phosphorylating, and hence inactivating, the growth-suppressing functions of Rb family proteins, thus promoting cell cycle progression. However, cdks also play a role in formation of growth-suppressing forms of pRb family molecules, including the "hypophosphorylated" species of pRb itself. Here, it is shown that normal human mammary epithelial cells have a high amount of cdk6 protein and activity, but all breast tumor-derived cell lines analyzed had reduced levels, with several having little or no cdk6. Immunohistochemical studies showed reduced levels of cdk6 in breast tumor cells as compared with normal breast tissue in vivo. Cdk6 levels in two breast tumor cell lines were restored to those characteristic of normal human mammary epithelial cells by DNA transfection. The cells had a reduced growth rate compared with parental tumor cells; cells that lost ectopic expression of cdk6 reverted to the faster growth rate of parental cells. Cell lines with restored cdk6 levels accumulated higher amounts of the Rb family protein p130 as well as E2F4, a suppressing member of the E2F family of transcription factors, in their nuclei. The results suggest that cdk6 restrains rather than stimulates breast epithelial cell proliferation and that its loss or down-regulation could play a role in breast tumor development.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
A complex network of proteins regulates the entry of cells into and through the cell cycle (1, 2). The Rb family proteins pRb, p107, and p130 play central roles in this network, primarily through binding to and regulating the E2F family of transcription factors, which control the expression of genes needed for cell cycle progression and metabolism, including DNA replication (3–6). The activities of Rb family proteins are regulated in part by phosphorylation performed by cyclin-dependent kinases (cdks; 7–12). The activities of cdks are themselves modulated by phosphorylation and by binding to cyclins and cdk inhibitors (CDKIs; 13, 14).

Defects in the expression, activity, or localization of members of the protein families that regulate Rb family proteins, have been observed in human tumors, including those originating in mammary epithelium. Alterations seen in significant numbers of breast tumors include overproduction of cyclin D1 (15, 16) or various isoforms of cyclin E (17–19), decreased production of the CDKIs p16Ink4a (20, 21) or p27Kip1 (22, 23), and a defect in the cytoplasmic/nuclear transport of p27Kip1 (24–26). Such alterations can result in defective regulation of Rb family protein function and inappropriate cell proliferation.

Control of Rb family protein activity involves regulation of abundance, phosphorylation, and other post-translational modifications, including acetylation (27–29). Rb family protein phosphorylation sites are of at least two types, which are preferentially phosphorylated by cyclin D-cdk4/cdk6 complexes or cyclin E/cdk2 (9–11, 30, 31). The differential functions of cdk4-, cdk6-, and cdk2-mediated phosphorylation of Rb family proteins remain controversial (see 32). For example, alternative models propose that cyclin D-cdk4/cdk6-mediated phosphorylation of pRb results either in the active, fully transcription-repressing, "hypophosphorylated" pRb (33) or in a partially repressing protein, which permits a low level of transcription of some E2F-responsive genes, such as cyclin E (27). Furthermore, whether cdk4 or cdk6 has a unique function in these models has not yet been adequately considered.

Activities of cdk4 and cdk6 are regulated by the same D-type cyclins and CDKIs (13, 14). Early analyses suggested that either of the two kinases predominated as the principle early G1-phase pRb kinase in various cell types (14, 34, 35). T lymphocytes were recognized as a rich source of active cdk6 (36, 37), while mammary epithelial cells appeared to have mostly cdk4 and little or no cdk6 (38). It appears likely that most normal cells have both kinases, but the ratio of the two differs greatly among cell types, and activities may be restricted to narrow regions of the cell cycle, increasing the difficulty of detection (37, 39–42). Differential timings in their activation throughout G1 phase suggested that cdk4 and cdk6 may each have unique functions. Distinct, complex effects of ectopic expression of cdk4 or cdk6 on the growth and differentiated states of several cell types, including mouse erythroleukemia cells and astrocytes, have also been described (43, 44). Because many of the proteins regulating cdk4/cdk6 are altered in breast tumors, it is important to determine the specific role of each kinase in mammary epithelial cell growth and function.

It is shown here that breast tumor cells generally have a reduced amount of cdk6 as compared with normal mammary epithelial cells. The results indicate that cdk6 restrains mammary epithelial cell proliferation, apparently through enhanced nuclear accumulation of p130 and E2F4. It has been proposed that p130 suppresses cell cycle progression in part by escorting the transcription factor E2F4 to the nucleus, where, in association with chromatin remodeling proteins, it represses transcription from E2F-regulated promoters (see 45). Loss of cdk6 and subsequent dysregulation of p130 would disrupt an important mechanism for restraining cell proliferation.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Comparison of cdk6 in Human Mammary Epithelial Cells and Breast Tumor-Derived Cell Lines
Human mammary epithelial cell (HMEC) strains are derived from normal breast epithelial cells and have a limited life span in vitro (46). Cellular contents of cyclins D1 and D3, cdk2, and cdk6 were compared by immunoblot analysis in growing HMECs and 10 tumor-derived cell lines (Fig. 1A ). Samples were prepared from equal cell numbers, and all four proteins were assayed using the same lysates. Quantitative analysis (Fig. 1D) showed that 5 of the 10 lines had more cyclin D1 than HMECs and 7 of the 10 lines overproduced cyclin D3 compared with normal cells. Most notable was the finding that all 10 tumor lines had less cdk6 than HMECs, with 7 having little or none. The same trend, especially the low cdk6 level in seven tumor lines, was seen if samples were normalized to protein content, indicated by actin levels, as shown in Fig. 1B. Next, cell extracts were prepared (using equal cell numbers), and cdk6 activity was measured using an in vitro kinase assay with truncated recombinant pRb protein as substrate (Fig. 1C). For HMECs and seven of the cell lines, there was a good correlation between cdk6 protein and enzyme activity (see Fig. 1D). Three cell lines (MCF-7, BT-20, and BT-474) had higher levels of enzyme activity than might be expected from cdk6 protein levels. Two of these lines (MCF-7 and BT-474) had elevated levels of both cyclins D1 and D3, which may have compensated for reduced cdk6 protein amounts. In Fig. 1C, kinase activity for an extract from human T cells is included (lane 12). HMECs (lane 1) have levels of cdk6 activity comparable with that of T cells. Two labeled bands are observed in the kinase reactions for some samples; only one represents phosphorylated pRb protein. As shown by comparison of lanes 10 and 13, the other protein (labeled "x") is not a form of pRb; it is detected when the reaction is performed with cdk6 from MDA-MB-157 cells without added pRb (lane 13) and appears to be a protein coimmunoprecipitated with cdk6.



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  		FIGURE 1. Breast tumor-derived cell lines have less cdk6 than normal HMECs. A. Normal HMECs and 10 tumor cell lines were maintained in a proliferative state and then harvested for assessment by immunoblotting of cyclins D1 and D3, cdk2, and cdk6. The cell types analyzed were HMECs (lane 1), MCF-7 (lane 2), BT-20 (lane 3), BT-474 (lane 4), BT-549 (lane 5), MDA-MB-453 (lane 6), MDA-MB-468 (lane 7), T-47D (lane 8), ZR-75-1 (lane 9), MDA-MB-157 (lane 10), and MDA-MB-134-VI* (lane 11). Samples derived from equal cell numbers were compared. B. Extracts from growing HMECs and the 10 tumor cell lines were assessed by immunoblotting for cdk6 and actin. The lanes were ordered to match that in A. C. The 11 cell types were assayed for cdk6 kinase activity. The lanes were ordered to match that in A, with the addition of lane 12, which shows the result of a kinase reaction with an extract from normal human T cells, and lane 13, which shows a kinase reaction performed with an extract of MDA-MB-157 cells without added pRb as substrate. The positions of the pRb protein used as a substrate and a protein (x), which was coimmunoprecipitated with cdk6 and phosphorylated in vitro, are marked. Analyses of all samples were performed at the same time; lanes were reordered to match that in A. D. A summary of cyclins D1 and D3, cdk2, and cdk6 protein levels and cdk6 kinase activity in HMECs and the tumor cell lines is shown. The results in A and C were quantitated as described in "Materials and Methods." Values for tumor cell lines are expressed relative to those for HMECs, which are set at 100. Kinase activities were quantitated using the pRb substrate band and not the coimmunoprecipitated band, which was observed with only some cell lines.

 
Immunohistochemical Detection of cdk6 in Normal and Tumor Breast Tissues
Changes in the levels of many molecules occur during establishment of cell lines. To examine if there was a differential accumulation of cdk6 in normal and tumor breast tissues in vivo, tissue sections were analyzed by an immunohistochemical assay. Sections from three breast tumors and corresponding normal tissue were examined in detail. Figure 2, A and B , shows typical normal breast tissue at two magnifications and illustrate a positive immunoperoxidase (brown) staining for cdk6. In normal tissue, staining was most prominent in epithelial cells lining the lumen of acini and ducts, although staining was also seen in underlying myoepithelial cells and isolated cells in the collagen matrix. Cdk6 appeared in the cytoplasm and nucleus of normal cells. Figure 2, C and D, shows regions of a tumor (from the same subject) having an appearance characteristic of many ductal carcinomas, with disordered tissue architecture and cell nuclei having abundant euchromatin. Cdk6 was present in cells but appeared to be largely excluded from nuclei. Other regions of the same tumor are shown in Fig. 2, E and F. Breast tumors often show great heterogeneity in appearance and content of prognostic indicators (47, 48). The cells in the upper part of Fig. 2E show little staining for cdk6; they have nuclei intensely stained (blue) by hematoxylin, as shown clearly at higher magnification (Fig. 2F). Cells in the lower part of Fig. 2E are arranged in a more normal architecture and many have cdk6 in cytoplasm and nuclei.



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  		FIGURE 2. Immunohistochemical detection of cdk6 in normal breast tissue and in breast tumor samples. Cdk6 levels were assessed using the immunohistochemical assay described in "Materials and Methods." Cdk6, shown by the brown staining, is seen in normal breast tissue (A and B) at two magnifications (using a 40x objective for A and a 100x oil immersion objective for B). Tumor tissue from the same individual is shown in C and E using the low-power objective and in D and F at high magnification. Sections were counterstained with hematoxylin.

 
Sections from 59 additional tumors representing a range of morphological types were examined. Cells were classified as having high, medium, or low levels of cdk6 in cytoplasm and nucleus. It was also considered that some tumors were heterogeneous in nature. Two tumors had substantial regions of cells that were clearly part of tumor tissue but had high cdk6 levels in both cytoplasm and nucleus. Both tumors were heterogeneous in appearance and had other regions composed of cells having low nuclear cdk6 levels and either medium (one tumor) or low (one tumor) cytoplasmic cdk6 levels. Twelve tumors had cells with medium levels of nuclear cdk6 and high levels of cytoplasmic cdk6. Nine of these 12 tumors were heterogeneous in appearance and had regions with low nuclear cdk6 levels and either high (seven tumors), medium (one tumor), or low (one tumor) cytoplasmic levels. It was notable that six tumors having cells with medium levels of nuclear cdk6 and high levels of cytoplasmic cdk6 had structures that most resembled normal acini and ducts. The remaining 43 tumors were composed primarily of cells with little nuclear cdk6 staining and either high (16 tumors), medium (17 tumors), or low (10 tumors) cytoplasmic cdk6. In summary, normal tissue showed a more positive staining for cdk6 than tumor tissue. As for breast tumor-derived cell lines, cdk6 levels varied among tumors. Cells in tumors showed a more marked decrease in cdk6 content in nuclei than cytoplasm as compared with normal cells. It should also be noted that examination of cdk6 in HMEC cell strains growing in culture showed the presence of cdk6 in both cytoplasm and nuclei, with a higher content in nuclei of most cells (data not shown).

Isolation and Characterization of Breast Tumor Cell Lines Overexpressing cdk6
To investigate the role of cdk6 in cell proliferation, cdk6 levels in two tumor cell lines (MDA-MB-453 and MDA-MB-468) were altered by transfection with plasmids encoding wild-type (wt) or dominant-negative (dn) cdk6. The two lines were transfected and clonal lines were established; a subset of lines was analyzed in detail. Figure 3A shows that five of six transfectants derived from MDA-MB-453 cells overproduced cdk6, with unchanged levels of cdk4 and cyclin D1. In lane 6 (Fig. 3A), analysis for cdk6 indicated that this wt cdk6 transfectant line did not overexpress cdk6. Initial analysis of this line after isolation showed that it had overproduced cdk6 (lane 8) compared with parental cells (lane 9) analyzed at the same time. Samples of this line when it was overproducing cdk6 (453 wt clone 6) and after it had lost ectopic cdk6 expression (453 wt clone 6rev) were used in subsequent analysis (see below). As shown in Fig. 3B, all transfected MDA-MB-468 cells produced more cdk6 than parental cells; levels of cdk4 and cyclin D1 remained relatively unchanged.



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  		FIGURE 3. Cell lines overexpressing wt cdk6 have a reduced growth rate compared with parental cells. The breast tumor cell lines MDA-MB-453 and MDA-MB-468 were transfected with the wt or dn form of cdk6, and clonal lines were selected in G418. After sufficient numbers of cells were obtained, they were transferred to growth medium without G418 and assessed for levels of cdk6, cdk4, and cyclin D1 protein by immunoblotting and for growth rate. A. The molecules were analyzed in parental MDA-MD-453 cells (lane 1), three dn cdk6 clonal lines (lanes 2, 3, and 4), and three wt cdk6 clonal lines (lanes 5, 6, and 7). As shown in lane 6, one clonal line did not overexpress cdk6 compared with parental cells. An extract of this cell line assessed at an earlier time after isolation and an extract of parental control cells harvested at the same time are shown in lanes 8 and 9, respectively. B. The contents of cdk6, cdk4, and cyclin D1 in parental MDA-MB-468 cells (lane 1), three dn cdk6 clonal lines (lanes 2, 3, and 4), and three wt cdk6 clonal lines (lanes 5, 6, and 7) are shown. C. Growth curves for the MDA-MB-453 cell lines examined in A were established by determination of cell numbers in cultures of proliferating cells at 24-h intervals for 4–5 days. Results are shown for the MDA-MB-453 tumor cell line ("453 parent" line), three dn cdk6 clonal lines ("dn" lines), and three wt cdk6 clonal lines ("wt" lines). These analyses was performed 1–2 weeks after cells were transferred out of G418 selection medium. Growth was also measured using the MDA-MB-453 cdk6 wt clone 6 cell line at a time when it had lost overexpression of cdk6 ("wt rev line" curve). D. Analysis of growth was performed with the MDA-MB-468 cell lines examined in B. Results are shown for the MDA-MB-468 tumor cell line ("468 parent" line), three dn cdk6 clonal lines ("dn" lines), and three wt cdk6 clonal lines ("wt" lines).

 
The growth rates of the cell lines were determined. As shown in Fig. 3C, the growth rates of the MDA-MB-453 parental line and three clonal lines expressing dn cdk6 were similar; the growth rates of the three lines overexpressing wt cdk6 were much reduced, by about 68% compared with the parental line. When this analysis was performed, 453 wt clone 6 line possessed a high content of cdk6 protein (Fig. 3A, lane 8). Subsequently, the growth rate was determined when the line was accumulating little cdk6 (Fig. 3A, lane 6). At this time, the growth rate of the line ("wt rev" line in Fig. 3C) was indistinguishable from that of the parental MDA-MB-453 cells. Loss of cdk6 was correlated with a return to the rapid growth rate of the parental tumor cell line. Analysis of MDA-MB-468 clones (Fig. 3D) showed that three lines overexpressing wt cdk6 had about a 70% reduction in growth compared with parental cells. Cell lines expressing dn cdk6 showed a modest reduction in growth, which appeared to be insignificant on repeated observation. Despite decreased growth rates, MDA-MB-453 and MDA-MB-468 cells transfected with wt cdk6 appeared morphologically identical to parental cells. Flow cytometric analysis showed no increase in apoptotic cells, as indicated by cells having a sub-G1 content of DNA after staining with propidium iodide. Distribution through the cell cycle was identical for parental and dn cdk6 transfectants. Analysis of wt cdk6 cells indicated that their greatly increased doubling times were due to increased transit times through all measured phases (G1, S, and G2) of the cell cycle (data not shown).

Altered p130 Content and Cellular Localization in Cells Overexpressing wt cdk6
It was reported that mouse 3T3 fibroblasts transfected with wt cdk6 had a decreased growth rate and overproduced p130 and p53 growth-suppressing proteins (42). These molecules were examined in the breast cell transfectants (Fig. 4A ). As in 3T3 cells, p130 accumulated to higher levels in breast cell lines overexpressing wt cdk6 as compared with parental cells or dn cdk6 transfectants. The p53 protein was not detected in MDA-MB-453 cells (49); in MDA-MB-468 cells, p53 levels were unchanged in wt and dn transfectants as compared with parental cells. The levels of two molecules reported to associate with p130, histone deacetylase (HDAC2) and transcription factor E2F4, were also unchanged in amount in transfected cells as compared with parental cells (Fig. 4A). Also shown (Fig. 4B) are results of analysis of pRb using two phosphorylation-specific antibodies, directed to pRbpT826 and pRbpSpT249/252, which are cyclin D/cdk-specific phosphorylation sites (30). No changes in the amount of the two phosphorylated species were detected in the transfectant MDA-MB-453 lines as compared with parental cells. It was also determined that overall amounts of pRb did not change in the MDA-MB-453 cells after transfection. As described (50), pRb was not detected in MDA-MB-468 cells (data not shown).



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  		FIGURE 4. Cell lines overexpressing cdk6 accumulate more p130 and have more p130 and E2F4 in their nuclei than parental cells. A. Immunoblot analysis for p130, p53, HDAC2, and E2F4 was performed using MDA-MB-453 parental cells (lane 1), dn cdk6 (lane2) and wt cdk6 (lane 3) transfectants, MDA-MB-468 parental cells (lane 4), and dn cdk6 (lane 5) and wt cdk6 (lane 6) transfectants. p, parental cell line; dn and wt, cell lines transfected with dn or wt cdk6, respectively. B. Immunoblot analysis for pRb (pT826), pRb (pSpT249/252), and Rb (total protein) was performed using MDA-MB-453 parental cells (lane 1) and dn cdk6 (lane 2) and wt cdk6 (lane 3) transfectants. C. Nuclei were isolated from MDA-MB-468 parental cells (p) and from cells transfected with dn cdk6 (dn) or wt cdk6 (wt). Nuclei were washed well and nuclear and cytoplasmic extracts were prepared. The contents of p130, E2F4, HDAC2, and actin in nuclear fractions and actin in the cytoplasmic fraction were examined by immunoblot analyses. D. Intracellular localization of p130 was determined by confocal immunofluorescence microscopy in parental MDA-MB-468 cells and in the dn cdk6 and wt cdk6 cell lines examined in A. Pictures were taken and processed at the same exposure levels and fluorescence intensity is thus indicative of p130 amount. E. The intracellular localization of E2F4 was determined by confocal immunofluorescence microscopy in parental MDA-MB-468 cells and in the dn cdk6 and wt cdk6 lines examined in A. Pictures were taken and processed at the same exposure levels and fluorescence intensity is thus indicative of E2F4 amount.

 
Next, the intracellular localization of p130 was examined. Nuclei were isolated from parental MDA-MB-468 cells and wt and dn cdk6 transfectants and probed for p130. As shown in Fig. 4C, wt cdk6 cells had more nuclear p130 than the other cell types. It has been suggested that p130 suppresses cell cycle progression in part by escorting E2F4, a transcription-repressing member of the E2F family, into the nucleus (see "Discussion"). Therefore, the nuclear contents of E2F4 in the three cell types were compared. As shown in Fig. 4C, wt cdk6 cells had more nuclear E2F4 than parental or dn cdk6 cells. The efficacy of the cell fractionation method was demonstrated by showing that nuclear fractions from the three cell types had equal amounts of HDAC2 (Fig. 4C), a predominantly nuclear protein that does not change in amount after transfection (see Fig. 4A). Because nuclei were washed well before extraction of nuclear proteins, they had a very low level of contamination by the cytoplasmic marker actin as compared with the amount of actin from cytoplasmic fractions prepared from the same numbers of cells (Fig. 4C).

The differential nuclear localizations of p130 and E2F4 were confirmed by confocal immunofluorescence microscopy. As shown in Fig. 4D, p130 was increased in overall amount (indicated by green fluorescence intensity) in MDA-MB-468 wt cdk6 transfectant cells as compared with parental and dn cdk6 cells. It also appeared that p130 accumulated to a greater degree in the nuclei of wt cdk6 cells than dn cdk6 cells. The amount of p130 in the nuclei of dn cdk6 transfectants was even less than that seen in the parent cells, suggesting that the dn form of cdk6 inhibited nuclear accumulation of p130. As shown in Fig. 4E, the level of E2F4 also was highest in the nuclei of wt cdk6 cells.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
In this report, it was shown that normal breast cells have relatively high amounts of cdk6 protein and activity, comparable with that seen in T lymphocytes. Breast tumor-derived cell lines analyzed had a reduced amount of cdk6, with some having little or none. The notion that mammary epithelial cells have little cdk6 may have arisen from examination of tumor cell lines or particular cell strains deficient in cdk6. We also observed differential levels of cdk6 in normal and tumor breast tissues in vivo. Even in areas of tumors, which had relatively high amounts of cdk6, its abundance appeared to be especially low in the nuclei, the site of action of cdks. The finding of high cdk6 levels in normal mammary epithelial cells is important as many defects seen in breast tumors are in proteins that interact not only with cdk4 but also with cdk6.

HMEC cultures have a limited life span in vitro and retain many of the characteristics of normal mammary epithelial cells (46). However, because they generally express markers characteristic of both basal and luminal epithelial cells, their value as an accurate model of normal breast epithelial cells remains controversial (see 51). Nonetheless, because a large portion of human breast tumors clearly originate in the ductal epithelium, they represent a convenient model for comparisons of "normal" and breast tumor cells. More informative are comparisons between primary normal and tumor tissue cells (as in Fig. 2), although definitive conclusions require examination of many samples.

Tumor cells engineered to express "normal" levels of cdk6 had a reduced growth rate compared with parental cells. Expression of a dn form of cdk6 had little impact on proliferation. Inhibition of proliferation by cdk6 was described previously in studies with mouse fibroblast 3T3 cells (42). These results were surprising as cdk6 has been considered a cdk4 homologue involved in cell cycle progression. For example, it was reported that enforced expression of cdk6, either alone (52) or with cyclin D1 (53), significantly shortened G1-phase length. However, these studies were performed with U2OS cells, which are defective in production of p16Ink4a (54), a protein that modulates cdk6/cdk4 activity. In other cases suggesting a stimulatory role for cdk6, a causal link between inhibition of cdk6 activity and inhibition of proliferation was not established. For example, studies with breast epithelial cells suggested that indole-3-carbinol inhibits cellular growth through a mechanism involving cdk6 suppression; however, a decrease in cdk2 activity was also observed (55). Indole-3-carbinol treatment of breast cancer cells also induces translocation of Bax to mitochondria and apoptotic cell death (56). Similarly, although inhibition of growth by vitamin D analogues or transforming growth factor-ß is accompanied by inhibition of cdk6 activity in some systems, cdk2 is also inhibited (57–59). Other studies taken as evidence of a growth-stimulatory role for cdk6 include findings of increased cdk6 amounts in some squamous cell carcinomas (60), gliomas (61), melanomas (62, 63), and malignancies of lymphoid origin (64). A contributory role for cdk6 in tumorigenesis has not been established in these systems; in many cases, other genes are amplified and/or overexpressed at comparable frequencies, such as cyclin D1 in melanoma (65).

When the transfection studies reported here were begun, it was surmised that ectopic expression of cdk6 might lead to sequestration of cyclin D, resulting in inhibition of cdk4 activity. This seems unlikely because phosphorylation of cyclin D kinase-directed sites on pRb appeared to be unchanged in cells expressing dn or wt cdk6. It was also noteworthy that inhibition of proliferation by cdk6 occurred in a cell line negative for pRb (MDA-MB-468). These results imply that suppression of proliferation was independent of cdk4 and pRb. The dn cdk6 used here, which is mutated in its ATP binding site, can, like wt cdk6, bind to D-type cyclins and CDKIs (66). It is likely then that the differential effects of wt and dn cdk6 on cellular proliferation are due at least in part to cdk6 enzymatic activity and not only to sequestration of cdk6 regulatory proteins. However, we have as yet found only small increases in cdk6 kinase activity in wt cdk6 transfectants (data not shown) in contrast to the large increases in cdk6 protein seen (Fig. 3). As described above, cdk6 activity apparently is restricted to only a narrow portion of the cell cycle and hence obscured when assaying randomly growing cultures. Alternatively, cdk6 may be inefficiently extracted from nuclei of cdk6 transfectants or inactivated during cell lysis, perhaps by the many CDKIs that are present in the cytoplasm of cdk6 transfected cell lines (unpublished results). These possibilities are currently under investigation.

It was shown that p130 levels and nuclear accumulation were altered by manipulation of cdk6 levels. An evolving model of cell cycle entry and G1 progression suggests that p130 plays a role in maintaining cells in G0, whereas pRb itself is functionally inert in G0 (9–11, 45, 67, 68). Presumably, formation of the phosphorylated species of p130 responsible for growth suppression would be accomplished at least in part by cyclin D-associated kinases (31, 69). Stimulation of cells to enter the cell cycle induces the synthesis of D-type cyclins (70, 71) and increased activation of cdk4/cdk6, which then alter further the phosphorylation status of p130 and pRb. Functions of p130 that maintain cells in the G0 state would be neutralized; conversely, pRb would be altered from its nonphosphorylated to "hypophosphorylated" state, an active E2F factor binding form that suppresses cell cycle progression (6, 9–11, 14). Cell cycle progression is then blocked by pRb, but many metabolic activities are induced, resulting in the near doubling of cell size, which occurs during G1 phase. In this model, cyclin D-cdk4/cdk6 would have several roles: maintaining cells in a resting state, stimulating cells to exit G0, but then blocking progression in G1 phase. G0/G1-phase regulatory phosphorylations of p130 and pRb are likely performed by a combination of cdk4 and cdk6, but the distinct functions of each kinase are unknown.

The mechanism of growth suppression by p130 can be at least 2-fold: facilitating the transport of E2F4 to the nucleus (45, 72–74) and directly binding and inhibiting cyclin/cdk2 (29, 75). The finding that cdk6 inhibition of cell proliferation might be mediated through p130/E2F4 interaction and nuclear localization is of interest in light of reports suggesting that some antitumor agents might act in part by similar mechanisms. Treatment of breast tumor cells with the pure estrogen antagonist ICI 182780 or the hybrid polar compound, suberoylanilide hydroxamic acid (an inhibitor of HDAC), also results in the accumulation of p130/E2F4 complexes (76, 77). Our observations indicate that the network of molecules involved in p130 regulation, which includes cdk6, might be a fruitful area of study in developing new strategies for treating breast cancers.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell Culture
Normal HMECs were from Clonetics/Cambrex (Walkersville, MD) and were maintained in mammary epithelial growth medium (MEGM) with bovine pituitary extract (52 µg/ml), human recombinant epidermal growth factor (0.01 µg/ml), insulin (5 µg/ml), hydrocortisone (0.5 µg/ml), gentamicin (50 µg/ml), and amphotericin B (50 µg/ml). Three independently isolated strains (Clonetics 2595, 4678, and 219) were used in the studies reported here. Cell lines were obtained from the American Type Culture Collection (Rockville, MD). BT-20 and MCF-7 lines were maintained in Eagle's MEM supplemented with 10% FCS. MCF-7 medium also contained 10 µg/ml insulin. BT-474, BT-549, T-47D, ZR-75-1, MDA-MB-157, MDA-MB-453, and MDA-MB-468 lines were grown in RPMI 1640 with 10% FCS. BT-474 and T-47D medium also contained 10 µg/ml insulin and BT-549 medium contained 1 µg/ml insulin. MDA-MB-134-VI* cells were maintained in RPMI 1640 with 20% FCS.

Immunohistochemistry
Tissue sections were deparaffinized and stained for cdk6 using an immunoperoxidase system with a primary antibody to cdk6 (C-21 rabbit polyclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA), a biotinylated secondary antibody, and an avidin/biotinylated horseradish peroxidase complex. All reagents, except primary antibody, were part of the Vectastain Elite ABC Kit (Vector Laboratories, Inc., Burlingame, CA) and the procedure was performed according to the manufacturer's instructions. 3,3'-Diaminobenzidine was used as substrate. Sections were counterstained with hematoxylin QS and mounted in VectaMount (Vector Laboratories). Matched normal/tumor breast sections were obtained from the University of Colorado Cancer Center. Additional tumor sections were purchased (product IMH-123/FH-AC1 Breast Cancer Histo-Array Slide) from Imgenex (San Diego, CA).

Immunoblot Analysis
Samples were prepared and analyzed as described previously (37, 42). Nuclei were isolated using a NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce Chemical Co., Rockford, IL) following protocols supplied by the manufacturer. Cellular contents of molecules were compared in samples prepared from equal cell numbers, unless noted otherwise. Reagents for the chemiluminescence immunoblotting detection method were from Amersham Pharmacia Biotech (Piscataway, NJ). Primary antibodies were from Santa Cruz Biotechnology, except the phosphorylation site-specific antibodies for pRb, which were from Biosource (Camarillo, CA). For quantitation, chemiluminescence results were scanned using an Epson (Long Beach, CA) 636 Scanner connected to a Macintosh G3 computer. Image files were imported into Adobe Photoshop 4.0 using Epson SilverFast software. Protein bands were quantitated using NIH Image 1.62 software.

Cdk6 Kinase Assay
Samples were prepared and the in vitro kinase assay was performed as described previously (36, 37). Recombinant p60Rb protein (QED/Canji, San Diego, CA) was used as substrate. After electrophoresis, gels were dried and labeled bands were detected by autoradiography (40). Quantitation of bands was accomplished as described above.

Cell Transfection
Cells were removed from culture vessels using trypsin, washed with complete medium, and suspended in 500 µl of complete medium (without antibiotics) containing 25 µg of transfecting DNA at a cell density of 4 x 106 cells/ml. Transfection was accomplished by electroporation (320 V) using a Cell-Porator (Invitrogen, Carlsbad, CA). Cells were permitted to recover overnight in growth medium. They were then seeded at low density in medium containing G418 (Geneticin, Life Technologies, Inc./Invitrogen, Carlsbad, CA) and maintained until colonies developed. Doses of G418 needed to kill nontransfected cells had been determined to be 300 µg/ml for MDA-MB-468 cells and 600 µg/ml for MDA-MB-453 cells. Colonies were isolated and grown to bulk cultures in G418. Duplicate cell samples were then maintained in medium without G418 for use in experiments. The wt and dn cdk6 plasmids (66) were gifts from E. Harlow, S. van den Heuvel, and J. LaBaer (Massachusetts General Hospital, Boston, MA). The preparations used here were sequenced to ensure their identities. It was confirmed that the pCMV cdk6 wt plasmid contained the previously reported cdk6 sequence (accession no. X66365) and that the pCMV cdk6 dn plasmid contained this sequence with a single base change from G to A in the ATP binding site of the kinase. This resulted in an alteration in amino acid sequence from Asp in the wt to Asn in the dn sequence (35, 66).

Confocal Immunofluorescence Microscopy
Immunofluorescence detection of molecules was performed using methods described previously (41, 78). Cells were fixed with freshly prepared paraformaldehyde, treated with 0.2% Triton X-100 to facilitate intranuclear labeling, incubated with the primary antibodies (10 µg/ml) described above, and incubated with FITC-tagged donkey anti-rabbit IgG (5 µg/ml). In most experiments, cells were also stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) to facilitate localization of nuclei.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Rhi-Hua Fan for technical assistance, Masayuki Nagasawa, Guiming Li, Paul Seligman, Martha R. Stampfer, and Ed Harlow for helpful discussions and reagents, and Hannah and Avi Kupfer for advice and assistance with confocal microscopy.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 Supported in part by grants from the Denver Metropolitan Chapter of the Susan G. Komen Breast Cancer Foundation and the Cancer League of Colorado, American Cancer Society (grant IM-746), NIH (grants HL-36577 and AI-42246), and University of Colorado Cancer Center (grant CA46934). Back

Received October 8, 2003; revised December 5, 2003; accepted January 12, 2004.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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