Retinoblastoma protein-overexpressing HL60 cells resistant ... - Nature

Oncogene (1998) 16, 2729 ± 2737  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc

Retinoblastoma protein-overexpressing HL60 cells resistant to 1,25-dihydroxyvitamin D3 display increased CDK2 and CDK6 activity and shortened G1 phase Qing Mei Wang, Xun Luo, Ahmed Kheir, Frederick D Coman and George P Studzinski Department of Pathology and Laboratory Medicine, UMD-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103, USA

Drug resistance that occurs during cancer chemotherapy has been a major problem in controlling neoplastic progression. To study the cellular mechanisms of acquired drug resistance we developed 1,25-dihydroxyvitamin D3 (1,25D3)-resistant sublines of promyelocytic leukemia HL60 cells which have increased proliferation rates (Exp. Cell Res., 224, 312, 1996; Cancer Res., 50, 5513, 1996). We report here that the resistant sublines display varying degrees of shortening of the G1 phase as compared to the parental HL60-G cells. Protein levels of cyclins E, D1, D2 and D3 are elevated in these resistant cell lines, and cyclin D1 is especially high in 40AF cells, which has the shortest G1 length. The protein levels of cyclin-dependent kinase (Cdk)2, Cdk4 and Cdk6 are not altered in the resistant sublines. Both Cdk2 and Cdk6associated kinase activites are increased in the resistant sublines, but not Cdk4 kinase activity. Protein levels of p27Kip1 are not consistently altered in the resistant sublines as compared to the parental HL60-G cells, but are reduced relative to HL60-G cells arrested by 96 h treatment with 1,25D3. Interestingly, the resistant cell lines constitutively express high levels of retinoblastoma protein (pRb), and pRb is highly phosphorylated, indicating that the G1 cyclin/Cdk complexes in the resistant cells are physiologically active. The results suggest that the increased activity of cyclin D/Cdk6, and perhaps cyclin E/Cdk2, lead to rapid hyperphosphorylation of pRb and consequently a shorter early G1 phase, and that in the resistant cells the increased ratio of cyclin E to p27Kip1 results in activation of Cdk2 and contributes to the abrogation of the 1,25D3-induced block to the S phase entry. Additionally, it is apparent that constitutively increased levels of pRb are compatible with increased rates of cell proliferation. Keywords: retinoblastoma; vitamin D; human leukemia; cell cycle; cyclins

Introduction Dierentiation therapy of human malignant diseases is being explored as a less toxic alternative or adjunct to chemotherapy (Warrel et al., 1991). In addition to retinoids (Castaigne et al., 1990; Tallman et al., 1997), 1,25-dihydroxyvitamin D3 (1,25D3) has potential as a dierentiating agent for the treatment of non-

Correspondence: GP Studzinski Received 16 October 1997; revised 19 December 1997; accepted 22 December 1997

lymphocytic leukemias (Studzinski et al., 1993), but its precise mode of dierentiating and anti-proliferative actions requires further elucidation before its clinical use is likely to be successful. Human promyelocytic leukemia HL60 cells cultured in vitro provide an extensively used model for mechanistic studies of the basis for the therapeutic actions of dierentiating agents (Gallagher et al., 1979; Studzinski et al., 1993). For instance, 1,25D3 has been shown to down-regulate the expression of the c-myc proto-oncogene (Reitsma et al., 1983; Brelvi and Studzinski, 1986) and up-regulate c-jun and c-fos genes (Muller et al., 1985; Kolla et al., 1994; Kovary and Bravo, 1991), apparently related to the antiproliferative and pro-dierentiation eects of 1,25D3 on these cells. Although it has been shown that these eects are separable (Zhang et al., 1994; Studzinski et al., 1997), are associated with modulation of the expression of several other genes such as mad (Wang et al., 1997b) and p27Kip1 (Wang et al., 1996; 1997a), and are believed to be mediated by the activation of the nuclear receptor VDR (Haussler and Norman, 1969; Holick et al., 1971), the details of how 1,25D3 controls cell proliferation remain to be elucidated. Several laboratories have shown that prolonged exposure of HL60 and other human leukemia cells to 1,25D3 produces resistance to both the growth-inhibitory and the dierentiation-inducing actions of 1,25D3 (Kuribayashi et al., 1983; Lasky et al., 1994). We have developed two independently derived series of sublines with increasing degrees of resistance to 1,25D3 (Wajchman et al., 1996; Studzinski et al., 1997) and found that the resistance to 1,25D3 is accompanied by markedly increased rate of cell proliferation. In the present study we investigated the potential mechanisms for this increase. We focused on the G1 phase since this is the most variable period in the cell cycle of mammalian cells (Pardee, 1989), and the cell cycle phase most aected by exposure of the parental HL60 cells to 1,25D3 (Godyn et al., 1994; Zhang et al., 1994). Our ®ndings show that the increased cellular levels cyclins D1 and D2 and higher ratios of cyclin E to p27Kip1, can explain the shortening of G1 in the 1,25D3-resistant HL60 cells, and that pRb, which is highly expressed in the resistant cells, is also markedly hyperphosphorylated.

Results Derivation of 1,25D3-resistant subline Human promyelocytic leukemia cells were incubated in the presence of increasing concentrations of 1,25D3 to

G1 cyclins and pRb in 1,25D3-resistant HL60 cells QM Wang et al

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which they developed gradual resistance. The steps of this procedure are summarized in Figure 1, and have been described in detail previously (Wajchman et al., 1996; Studzinski et al., 1997). Shortening of G1 traverse in resistant sublines To investigate the cell cycle transit of the 1,25D3 resistant sublines, G1 length was determined using two methods. In the ®rst method the cells were arrested in G2/M phase by 4 mg/ml of nocodazole, an inhibitor of mitosis (Sladek and Jacobberger, 1992; Yang and Sladek, 1995). Since following the addition of nocodazole there should be no cells entering the G1 phase, the time required for 50% of the cells to leave the G1 phase (half-time of cell residence in G1) is proportional to the G1 length. Using ¯ow cytometry, we determined the percentage of cells in G1 phase at various time points (0, 2, 4, 6 and 8 h) after addition of nocodazole. The time points were plotted on a semilogarithmic scale (Figure 2) and the exponential regression equation (y=ke7lt) was derived (see Materials and methods section). The half-time of cell residence in G1 was calculated according to the exponential regression equation. As shown in Table 1, resistant sublines displayed shorter G1 phase than the HL60-G cells, with 40AF cells requiring the shortest time to `half-empty' the G1 compartment. Several additional sublines were also used in these experiments, and as summarized in Table 1 the

shortening of G1 phase was found in each subline. We con®rmed the order of relative lengths of the G1 transit times using another method. The cell number and the percentage of cells in the G1 were determined at 24, 48, 72 and 96 h and the doubling time was derived by semilogarithmically plotting cell number versus time. The G1 lengths were then calculated using

Figure 2 An example of half-time of cell residence in the G1 compartment. HL60-G, 40AF, 40B, 200B cells were treated with nocodazole (4 mg/ml) for 2, 4, 6 and 8 h. The percentage of cells in the G1 phase at each time point was determined by ¯ow cytometry, and these values were plotted using a logarithmical axis versus time in the presence of nocodazole on an arithmetic axis. The regression coecient (r2) was 50.92 in all experiments

Figure 1 Development of 1,25D3-resistant HL60 cell sublines, designated series A and series B. Promyelocytic leukemia cell subline HL60-G was cloned out from HL60-240 cells obtained from ATCC (Bethesda, MD), and cultured in increasing concentrations of 1,25D3. Series A sublines were derived by passaging only the non-adherent cells, and series B sublines were derived by passaging both the non-adherent and the adherent cells. The sublines were designated according to the highest nanomolar concentration of 1,25D3 in which the cells were grown


the formula provided in the Material and methods. As shown in Table 1, the G1 transit times derived by both methods were shorter in the resistant sublines than in the HL60-G cells. While the lengths of the G1 phase for any one cell line were dierent when measured by these two techniques, perhaps due to the secondary eects of nocadozole, G1 lengths in 1,25D3 resistant cells were always shorter than G1 lengths in 1,25D3susceptible cells. Absence of cyclin A in the G1 phase of HL60 cells The initial timing of the appearance of cyclin A in the cell cycle is a subject of controversy and may depend on the cell type. For instance, cyclin A was found in the late G1 phase by Dou et al. (1993), whereas in other studies cyclin A was detected only in the S phase and G2 phase (Pagano et al., 1992; Gong et al., 1995). In order to assess if cyclin A activation of Cdks may play a role in the traverse of G1 phase by HL60 cells, we determined the cell cycle timing of the expression of cyclin A in our system (Figure 3). Logarithmically growing HL60 cells were elutriated centrifugally, and three fractions of HL60 cells were collected. As shown by ¯ow cytometry (Figure 3a), in parental HL60-G cells fraction 1 consisted of 81% G1 cells, and 17% S phase cells; fraction 2 was composed mainly of S phase cells (68%) and smaller percentages of G1 phase (26%) and G2 phase (6%) cells; fraction 3 contained 61% S phase cells, 34% G2 cells and only 5% G1 phase cells. We examined cyclin A levels in these three fractions by Western blot analysis. The blot in Figure 3a shows that fraction 1 which was composed mainly of G1 cells had no immunodetectable cyclin A; however, fractions 2 and 3 which had only a small proportion of G1 cells and higher amounts of S and G2 cells had increasingly higher levels of cyclin A. Similar results were obtained when the resistant sublines were examined, although the amounts of cells sucient for Western analysis were obtained in only two cell cycle fractions of each subline; for instance, fraction 1 of 40B cells which was essentially pure G1 fraction had no detectable cyclin A, whereas fraction 1 in 200B cells, which had 84% of G1 cells and 16% S phase cells, showed a weak cyclin A signal (Figure 3b).These results demonstrate that in HL60 cells, both parental and 1,25D3-resistant, cyclin A is expressed in the G2 phase and in the S phase, but not in the G1 phase, and is therefore unlikely to in¯uence the G1 phase traverse. Upregulation of G1 cyclins in resistant sublines To investigate the mechanisms underlying the shortening of the G1 phase in 1,25D3-resistant cell sublines

we examined the levels of p27Kip1, shown to be the principal mediator of G1 block in this system (Wang et al., 1996; 1997a), and of G1 cyclins, which are positive regulators of the G1-S phase transition (Sherr, 1993). Cyclins D1, D2 and D3 are known to peak in the mid-G1, whereas cyclin E accumulates in the late G1 phase with a peak at the G1/S transition (Baldin et al., 1993; Quelle et al., 1993; Ohtsubo et al., 1995). Cyclins D and E all bind and activate the Cdks, which provide the driving force for cell cycle progression at this stage. As shown in Figure 4a, the marked up-regulation of p27Kip1 by a 96 h exposure to 1,25D3 became attenuated upon prolonged exposure to 1,25D3 at a range of concentrations from 20 nM (20AF cells) to 200 nM (200 B cells). The protein levels of D cyclins were also increased by a 96 h exposure to 1,25D3, and in the case of cyclins D1 and D2 this persisted when the cells developed resistance to 1,25D3 (Figure 4b and c). In contrast, cyclin D3 levels in the resistant sublines were essentially the same as in the parental cells (Figure 4d). Yet another situation was seen when protein levels of cyclin E were determined; short exposure to 1,25D3 reduced cyclin E levels, but there was a marked increase in the largest cyclin E isoform in the resistant sublines (Figure 4e), resulting in higher cyclin E to p27Kip1 ratios (Table 2). In contrast to the marked increases in G1 cyclin protein levels, the expression of Cdks was not found to be appreciably altered in the 1,25D3-resistant sublines (Figure 5). Short-term exposure of the parental cells to 1,25D3 down-regulated the levels of Cdk4 and Cdk6, but the levels returned to parental levels in most of the resistant sublines. Cdk2 did not appear to be regulated by 1,25D3 at all (Figure 5). Activity of cyclin dependent kinases Cdk2 kinase activity was determined by phosphorylation of histone H1, one of its principal substrates (Tsai et al., 1991). As shown in Figure 6a, most of the 1,25D3-resistant sublines tested (40AF, 100AF, 40B) had higher Cdk2 kinase activity than HL60-G, whereas HL60-G cells treated with 1,25D3 had very low Cdk2 kinase activity. The 200B cells, which partially reverted to the parental phenotype and express some differentiation markers (Studzinski et al., 1997), had Cdk2 kinase activity similar to the parental cells (Figure 6a). Since the traverse of the restriction (R) point in the G1 phase (Pardee, 1974) is thought to be largely controlled by phosphorylation of pRb by Cdk4 and/or Cdk6 combined with one of the D type cyclins (reviewed by Hunter and Pines, 1994), Cdk4 and

Table 1 Comparison of two methods for estimating G1 length in parental and 1,25D3-resistant sublines Half time of cell residence in G1 Determined by nocodazole h % of HL60-G HL60-G 30A 30AF 40AF 100AF 40B 200B

18.1+6.4 11.2+3.5 7.2+1.8 2.9+0.3 6.5+4.9 6.1+3.5 6.7+3.5

100 62 40 16 36 33 37

G1 length Determined by doubling time h % of HL60-G 26.5+1.6 11.0+0.1 13.3+1.3 7.2+0.2 9.9+0.9 18.8+2.1 20.9+3.0

100 42 50 27 37 71 79

2731


2732

a

1

2

3

Cyclin A

b

40B Fractions

1

200B 2

1

2

Cyclin A

Control Figure 3 Absence of cyclin A in G1 phase in parental and 1,25D3-resistant HL60 cells. (a) HL60-G cells were elutriated centrifugally into three fractions as described in Materials and methods. Each elutriation fraction was analysed by ¯ow cytometry to determine the cell cycle stage. Total protein was extracted from cells in each elutriation fraction and cyclin A protein levels were determined by Western blotting. Lane 1=Fraction l; Lane 2=Fraction 2; Lane 3=Fraction 3. Densitometric quantitation of the Western blot for cyclin A was performed using image analysis software (NIH Image). (b) 1,25D3-resistant HL60 cell sublines (40B and 200B) were elutriated into four fractions, which were analysed by Flow Cytometry of PI-stained cells. Sucient cells were present in two fractions to determine cyclin A protein levels by Western blotting. To demonstrate the equal loading, the blot used for cyclin A determination was reprobed with anti-calreticulin antibody, which shows little change in expression in nondierentiating HL60 cells


200B

100B

50B

40B

100AF

40AF

30AF

30A

GD3

20AF

GC GC

a

200BW

Cdk6 kinase assays were performed using pRb as the phosphorylation substrate. As shown in Figure 6, Cdk6 activity was higher in the resistant sublines (40AF, 40B) than in the HL60-G cells and lower in the HL60G cells treated with 1,25D3. However, the barely detectable Rb phosphorylation activity of Cdk4

P27

GD3

20AF

30A

30AF

40AF

100AF

40B

50B

100B

200B

200BW

GD3

20AF

30A

30AF

40AF

100AF

40B

50B

100B

200B

200BW

b

Cyclin D1

GC

c

showed no such increases in the resistant sublines, though this activity paradoxically increased in cells exposed to with 1,25D3 for only 96 h (Figure 6b), as reported previously (Wang et al., 1997a). Alterations in Rb expression and phosphorylation The increased in vitro Cdk6 kinase activity that correlates with elevated levels of cyclins D1 and D2, and with the shortening of the G1 phase suggests that the Cdk6/cyclin D complexes accelerate progress through the G1 phase by phosphorylating pRb in vivo. Figure 7 shows that this indeed is the case. Each subline showed predominantly hyperphosphorylated pRb (ppRb), in contrast to parental cells exposed to 1,25D3 for only 96 h, where only hypophosphorylated pRb was found (Figure 7). The resistant sublines also had much higher protein levels of total pRb than the parental cells. Densitometric measurements showed that the sublines had 3 ± 10-fold increased protein levels compared to pRb levels in parental cells. This was not due to the increased ploidy in some of these sublines (Wajchman et al., 1996), since the near-diploid sublines, e.g. 20AF and 40B, had total pRb levels similar to the levels in near-tetraploid sublines e.g., 40AF and 100B. Thus, the resistant sublines characteristically express high levels of hyperphosphorylated pRb.

Cyclin D2

Discussion

200BW

200B

100B

50B

40B

100AF

40AF

30AF

30A

20AF

GC

Cyclin D3

GD3

This study provides a mechanism for the development of resistance to the growth inhibitory and differentia-

200BW

200B

100B

50B

40B

100AF

40AF

30AF

30A

GD3

GC

d

200BW

200B

100B

50B

40B

100AF

40AF

30AF

30A

GD3

GC

e Cdk2

Cyclin E

Figure 4 Western blot analysis of p27Kip1 and G1 cyclins in the indicated 1,25D3, -resistant HL60 cell sublines. (a) Protein levels of p27 in untreated HL60-G cells, HL60-G cells treated with 1,25D3 (161077 M) for 96 h, and sublines 20AF, 30A, 30AF, 40AF, 100AF, 40B, 50B, 100B, 200B and 200BW. (b) Protein levels of cyclin D1 in cells shown in a. (c) Protein levels of cyclin D2 in cells shown in a. (d) Protein levels of D3 in untreated HL60-G cells, HL60-G cells treated with 1,25D3 (161077 M) for 96 h, and resistant sublines (30A, 30AF, 40AF, 100AF, 40B, 50B, 100B, 200B and 200BW). (e) Protein levels of cyclin E in cells shown in d. Equal protein content in each lane was con®rmed by Ponceau S staining before immunoblotting

Cdk4

Cdk6

Figure 5 Protein levels of Cdks determined by Western blotting in 1,25D3-resistant HL60 sublines. Immunoblots for Cdk2, Cdk4 and Cdk6 of cell extracts shown in Figure 4a

Table 2 Cyclin E to p27Kip1 ratios GC

GD3

30A

30AF

40AF

100AF

40B

50B

100B

200B

200BW

Cyc E 152+10.1 86+3.3 2263+34.4 2709+37.4 2384+34.4 1712+27.9 2578+35.1 2990+39.1 2592+36.7 791+20.4 1657+30.7 p27 31+2.8 4307+0.7 22+0.7 16+15.6 99+82.1 109+76.4 23+30.5 151+5.6 34+12.1 15+8.5 26+9.9 Cyc E/p27 4.9 0.02 100.6 169.3 24.1 15.7 110.2 199.4 76.2 52.7 63.7 Protein levels were quantitated using image analysis software. The values are shown in arbitrary units+s.e. of three determinations

2733


2734

100B

200B

200BW

100B

200B

200BW

50B

40B

100AF

40AF

30AF

30A

GD3

GC ppRb

GC

200B

40B

100AF

40AF

GD3

IP: anti-Cdk2 Ab

GC

a

Beads only

a

pRb Histone H1

50B

40B

100AF

40AF

30AF

30A

GC

40B

40AF

D3

GC

IP: anti-Cdk6 Ab

c

Beads only

GST-pRb

c

GD3

GC

40B

40AF

D3

GC

IP: anti-Cdk4 Ab

Beads only

b

GC

b

GST-pRb Figure 6 Cdk2, 4 and 6-associated kinase activity. (a) Cdk2associated kinase activity. Three hundred micrograms of total lysate from untreated HL60 cells and HL60-G cells treated with 100 nM 1,25D3 for 96 h, or from 40AF, 100AF, 40B, 200B cells, were immunoprecipitated with anti-Cdk2 antibody, and histone H1 was used as the phosphorylation substrate. (b) Cdk4associated kinase activity. Total lysates prepared from untreated HL60 cells and HL60-G cells treated with 100 nM 1,25D3 for 96 h, 40AF, and 40B cells were immunoprecipitated with antiCdk4 antibody. GST-pRb was used the phosphorylation substrate. (c) Total lysates prepared from cells as shown in b were immunoprecipitated with anti-Cdk6 antibody. GST-pRb was used as the phosphorylation substrate. In all the kinase assays, beads without addition of the antibody were used as controls

tion promoting eects of 1,25D3, and demonstrates several unexpected features of this complex differentiation system. Particularly interesting is the ®nding that high cellular levels of Rb protein are compatible with rapid rates of cell proliferation, and appear to be associated with an enhanced cyclin-dependent kinase activity. Arrest of cell cycle progression in dierentiating cells has been recognized for a considerable time (Baserga, 1985), but the mechanisms are only recently becoming elucidated. For instance, a threshold ratio of cyclin E to p27Kip1 has been reported to be essential for the G1 to S phase transition in several cell systems (Polyak et al., 1994; Nourse et al., 1994; Coats et al., 1996). Thus, the ®nding here that the levels of cyclin E are considerably increased in the resistant sublines compared to the parental HL60-G cells, untreated or treated for 96 h with 1,25D3, together with failure to detect a consistent increase in p27Kip1 levels, provide a potential explanation for the loss of the G1 to S phase block in the resistant sublines. This correlates with the

Figure 7 pRb expression and phosphorylation. (a) The pRb protein levels were determined in untreated HL60-G cells, HL60G cells treated with 1,25D3 (161077 M) for 96 h, and the resistant sublines (30A, 30AF, 40AF, 100AF, 40B, 50B, 100B, 200B and 200BW) using Western blots. The hyperphosphorylated (ppRb) and hypophosphorylated (pRb) proteins were identi®ed by their mobility on the SDS ± PAGE gel. (b) Ponceau S staining of total proteins. C. Ratio of Rb protein (pRb and ppRb) levels to total proteins. The intensity of the signal was determined using image analysis software. The ratios are shown in arbitrary units of three determinations

recovery of the ability of the resistant cells to phosphorylate histone H1 by Cdk2 (Figure 6a), that appears to be required for DNA replication in the S phase. Since, as noted above, cyclin A in this system is virtually absent in the G1 and early S phase (Figure 3), and levels of Cdk2 appear essentially invariant in this system (Figure 5), the increased levels of cyclin E are likely to control the Cdk2 activity and consequently the entry from G1 into the S phase. In addition to the development of resistance to 1,25D3, the sublines exhibit increased proliferation rates (Wajchman et al., 1996; Studzinski et al., 1997), mainly due to shortened G1 phase (Figure 2 and Table 1). There is some variability between the dierent sublines in this regard, but in general series A cells have shorter G1 phases than the B series cells. This correlates best with cyclin D1 levels (Figure 4), consistent with the reports that ectopic expression of this cyclin in rodent cells results in more rapid transit of the G1 phase (Quelle et al., 1993; Jiang et al, 1993). However, increased levels of cyclins D2 and E are also present (Figure 4), suggesting that several G1 cyclins together contribute to the more rapid transition of the R point, presumably by phosphorylating pRb (Weinberg, 1995). This is consistent with previous observa-


tions that cyclins D1 and E control dierent ratelimiting events in the cell cycle traverse (Resnitzky and Reed, 1995). However, although cyclin D3 was noted to be the D-type cyclin most commonly expressed in dierent cell types (Juan et al., 1996), cyclin D3 does not appear to be involved in the control of G1 traverse in the cells studied here (Figure 4). Increased levels of cyclins D1 and D2 correlate with the increased in vitro pRb phosphorylating activity of Cdk6, but Cdk4 does not show increased activity in the resistant sublines (Figure 6), though, as reported previously (Wang et al., 1997a), short term 1,25D3 treatment paradoxically increased Cdk4 activity and was paralleled by increases in the levels of all three Dtype cyclins. We have suggested (Wang et al., 1997a) that this phenomenon is related to the potential for resumption of replicative activity in 1,25D3-treated HL60 cells and may be evocative of its physiological counterpart, the maturation of macrophages, which can reenter the cell cycle after prolonged quiescence (Hume and Gordon, 1982; Sawyer, 1986). The increased phosphorylation of pRb by Cdk6 determined in vitro is strikingly paralleled by extensive phosphorylation of pRb in resistant sublines analysed by Western blotting (Figure 7). Since pRb is thought to be phosphorylated in all stages of the cell cycle except the early, pre-restriction point, segment of the G1 (Weinberg, 1995), we interpret this ®nding as due to a very rapid phosphorylation of the newly synthesized pRb by cyclin D-activated kinase activity, and thus a greatly shortened early G1 phase. Interestingly, the resistant sublines were found to exhibit markedly increased expression of Rb, present in the hyperphosphorylated form. pRb is generally involved in downregulation of S phase-speci®c genes, and is thus considered a negative regulator of cell growth (Weinberg, 1995). Binding and sequestration of E2F transcription factors by Rb, and by the related proteins pl30 and pl07 is well known (Shirodkar et al., 1992; Cobrinik et al., 1993), but other mechanisms of pRb action on growth control are also becoming apparent. For instance, binding of pRb to E2F can convert it into an active repressor of transcription (Weintraub et al., 1995). Even more interestingly, the transcription of several genes has been reported to be stimulated by Rb, including cyclin D1 (Muller et al., l994). This is probably mediated by binding of pRb to transcription factors other than E2F, and thus has an indirect eect on transcription (Sanchez and Dynlach, 1996). For instance, binding of pRb to the glucocorticoid receptor has been reported to promote transcription by targeting a putative helicase (hBrm) to the glucocorticoid receptor promoter and thereby assist in chromatin reorganization through nucleosomal disruption at the promoter (Singh et al., 1995). Also, the eect of pRb appears to be dependent on the cellular context, since the regulation of the TGF-beta 1 gene promoter by pRb can be either positive or negative, depending on the cell type (Kim et al., 1991). These ®ndings raise the possibility that the increased expression of pRb in the resistant sublines is also related to the accelerated transit of these cells through the G1 phase by activating a subset of growth-related genes. Alternatively, the role of pRb may be to inhibit phenotypic dierentiation, since ectopic overexpression of pRb resulted in a block to 1,25D3 -induced dierentiation in

a related monocytic leukemia cell line, U937 (Bergh et al., 1997). Notably, in our series of sublines the increased pRb levels were found in the earliest subline derived from the wild-type HL60-G cells (20AF), and the subline that partially displays the monocytic markers (200B) (Studzinski et al., 1997) had somewhat lower levels of pRb than the other sublines (Figure 7). Elevated levels of pRb may therefore be responsible for the undierentiated phenotype of HL60 cells after long-term growth in the presence of 1,25D3. A potential mechanism for upregulation of pRb expression in HL60-G cells propagated in the presence of 1,25D3 is provided by the presence of Sp1 sites in the human Rb promoter (Kim et al., 1992; Chen et al., 1994), since the 1,25D3-resistant sublines have recently been shown to have increased DNA-binding by Sp1 (Studzinski et al., 1997). This link is speculative but warrants further studies of the complex relationships between growth regulation and pRb expression. The identi®cation of human cells with high endogenous expression of pRb should aid investigations of both negative and positive control of cell proliferation and dierentiation by pRb.

Materials and methods Cell culture Human promyelocytic leukemia HL60-G cells were grown in RPMI 1640 medium (Mediatech, Washington, DC) supplemented with 10% heat-inactivated BCS (Hyclone Laboratories, Logan, UT), and 1% L-glutamine (Mediatech) at 378C in an environment of 5% CO2. Derivation of 1,25D3-resistant HL60-G sublines has been shown in Figure 1. These cultures were further supplemented with nanomolar concentrations of 1,25D3 corresponding to the numerical designation of the subline (e.g., 40AF=40 nM). The letter F was added to the designation when cells were recovered from frozen stocks. Cell viability was determined by trypan blue (0.4%) exclusion. Cell cycle distribution Three million cells were washed in 16PBS and incubated for 2 h with propidium iodide (10 mg/ml, containing 0.1 M sodium citrate and 0.1 Triton X-100) at 48C. The DNA content was determined by Epics Pro®le II ¯ow cytometer (Coulter Electronics, Hialeah,FL), and analysed using the Multicycle Software Package (Phoenix Flow Systems, San Diego,CA). Determination of the length of the G1 phase HL60-G cells and representative 1,25D 3-resistant cell sublines (30A, 30AF, 40AF, 100AF, 40B, 200B) were seeded at 200 K/ml and treated with nocodazole (4 mg/ml) for 2, 4, 6 and 8 h. Cells at the zero time point were used as controls. Percentage of cells in the G1 phase at each time point was determined by ¯ow cytometry, and plotted semilogarithmically versus the duration of nocodazole treatment, and the exponential regression equation y=k e7lt was derived (y is percentage of cells in G1, t is the duration of nocodazole administration, k and l are constants). The half-time of cell residence in G1, which is the time between addition of nocodazole and 50% of G1 cells emptying the G1 phase, was calculated as t1/2=0.693/ l. The G1 length was also estimated by determining the doubling time and the proportion of cells in the G1 phase. The cell number was determined at 0, 24, 48, 72 and 96 h

2735


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in culture and plotted semilogarithmically versus the time, and the doubling time was calculated from the slope of the straight line thus obtained. The proportion of cells in the G1 phase was derived from the average percentage of cells in the G1 phase, measured by ¯ow cytometry. The length of the G1 phase was calculated using the following formula: TG1/Tc=1n (FG1+1)/ln 2 (TC is the doubling time, TG1 is the duration of G1 and FG1 is the fraction of cells in G1). Centrifugal elutriation of HL60 cells A Beckman J6 elutriation rotor was washed with 250 ml of 70% ethanol followed by 250 ml of elutriation buer (PBS containing 2 mM EDTA). The centrifuge was started and the rotor was maintained at 2700 r.p.m. and 188C during the entire procedure. HL60 cells (4.56107 cells per condition) were harvested, resuspended in 10 ml elutriation buer, and loaded onto the elutriation rotor at a pump speed of 18 ml/min. One hundred and ®fty ml fractions were collected at pump speeds of 30, 38, 50 and 70 ml/min. Each fraction was pelleted and resuspended in 3 ml of chilled PBS-EDTA. Aliquots were removed for cell counting and ¯ow cytometric analysis, the remaining cells were pelleted and solubilized, and the proteins extracted for Western blot analysis.

Biotechnology Inc. (UBI), Lake Placid, NY), Cdk4 (UBI), Cdk6 (Pharmingen, San Diego, CA), and p27 (UBI) were applied at optimized concentrations. The proteins were visualized using a chemiluminescence assay system (Amersham Life Science Inc., Arlington Heights, Illinois), and quantitated by image analysis software (NIH Image). Kinase activity assays Total lysate was incubated with anti-Cdk2, anti-Cdk4 and anti-Cdk6 polyclonal antibodies for 2 h and then protein-A agarose beads (Gibco BRL, Gaithersburg, MD) for 1 h. The beads were then washed three times in lysis buer and twice in kinase buer (50 nM HEPES, 10 mM MgCl2, 5 mM MnCl2, 1 mM DTT, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 10 mg/ml pepstatin A, 0.2 mM PMSF, 20 mM NaF, 0.2 mM NaV04). The beads were then incubated for 30 min with 40 mL kinase reaction buer (10 mM ATP, 0.4 mCi/ml [g32P]ATP, 40 mg substrate/ml). Histone H1 (Boehringer Mannheim Co., Indianapolis, IN) was used as kinase substrate for Cdk2, and GST-pRb (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was used as kinase substrate for Cdk4 and Cdk6. The reaction was stopped by adding 36SDS sample buer (150 mM Tris, 30% glycerol, 3% SDS, 15 mg/ml bromophenol blue dye, 100 mM DTT). The supernatant was separated on an SDS ± PAGE gel and the radioactivity was detected by autoradiography.

Western blotting Total cell lysates were obtained by exposing 306106 cells to lysis buer (20 mM Tris-HCl at pH 7.4, 0.25 M sucrose, 1 mM phenylmethylsulfonyl ¯uoride, 2 mM EDTA, 10 mM EGTA, 10 mg/ml leupeptin, 10 mg/ml aprotinin and 5 mg/ ml pepstatin A). Protein concentrations were determined using Bio Rad Kit (Bio-Rad Laboratories, Hercules, CA). An aliquot of 30 mg of total protein was separated on 13% SDS ± PAGE gel and transferred to a nitrocellulose membrane. Ponceau S staining was utilized to verify that equal amount of protein was present in each lane. Primary antibodies against cyclin E (Oncogene Science Inc., Cambridge, MA), cyclin D1 (Oncogene), Cdk2 (Upstate

Abbreviations BCS, bovine calf serum; Cdk, cyclin-dependent kinase; 1,25D3, 1,25-dihydroxyvitamin D3; PMSF, phenylmethylsulfonyl ¯uoride; pRb, retinobastoma protein. Acknowledgements We are grateful to Dr Milan Uskokovic (HomannLaRoche, Nutley, NJ) for the gift of 1,25D3. This study was supported by USPHS grant RO1-44722 from the National Cancer Institute.

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