Hypophosphorylation of the RB Protein in S and G2 as Well as G1 during Growth Arrest

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

241, 324–331 (1998)

EX984007

Hypophosphorylation of the RB Protein in S and G2 as Well as G1 during Growth Arrest1 Andrew Yen2 and Rhonda Sturgill Cancer Biology Laboratory, Department of Pathology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

The RB tumor suppressor protein is a cell cycle regulator, where hypophosphorylated RB is associated with G1/0 arrest and its cyclin-dependent phosphorylation in G1 allows progression from G1 to S. The present report shows that in human leukemia cells induced to undergo growth arrest with sodium butyrate or DMSO, hypophosphorylation of the RB protein is not G1 restricted and also occurs in S and G2/M cells as well as in G1 cells when growth is inhibited. While all of the RB protein in G1/0 cells is hypophosphorylated, residual cells in S and G2 have significant detectable amounts of hypophosphorylated RB as well as still hyperphosphorylated RB protein. Thus RB hypophosphorylation can be induced in S and G2 as well as the G1 phase. The results show that growth retardation in other than the G1 phase is associated with occurrence of hypophosphorylated RB. RB may thus have a broader capability to inhibit proliferation than just in G1. q 1998 Academic Press

INTRODUCTION

The RB (retinoblastoma tumor suppressor) gene is the tumor suppressor gene whose biallelic loss of function confers susceptibility to retinoblastoma and other neoplasias [1–3 reviews]. RB encodes a 105-kDa nuclear phosphoprotein containing a nuclear translocation sequence and a leucine zipper and is a putative proximal regulator of cell cycle progression. RB can be phosphorylated by cyclin-dependent kinases. Cyclin D-, E-, or A-dependent kinases, for example, can use RB as a substrate. The hypophosphorylated RB protein binds viral antigens that promote DNA synthesis and transcription factors needed to induce S phase specific genes, most notably members of the E2F family. In the presently prevalent paradigm of RB function, as cells transit the cell cycle, phosphorylation of hypophosphorylated RB—possibly at the G1 restriction point [4]— by cyclin-dependent kinases [5–8] causes the release 1 Supported in parts by grants from the USPHS (NIH) and the USDA. 2 To whom correspondence and reprint requests should be addressed.

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0014-4827/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.

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of transcription factors that selectively bind hypophosphorylated RB. These transcription factors, the prototype of which is the E2F family, are thereby liberated to target E2F site containing promoters and transcriptionally activate genes needed for cell cycle progression to S phase. For example, thymidine kinase, thymidilate synthetase, dihydrofolate reductase, and ribonucleotide reductase represent such instances of E2F-dependent transcriptional activation. While this paradigm enjoyed significant early success, more recent data suggest that the model is limited and there is potentially a broader role for RB function. A variety of nonconcordances with this model have been reported in relationships between expression of cyclins and RB phosphorylation [9–11] as well as between RB phosphorylation and the cell cycle phases [12–14]. Nor does the relationship between RB phosphorylation and availability of free E2F necessarily correspond to the anticipations of this paradigm [15]. Certain recent data suggest the possibility that RB may have a more global regulatory role influencing general levels of cellular metabolic activity. RB has been found to be concentrated in nucleoli [16], the site of transcriptional activity. Hypophosphorylated RB binds hBRG1 and hBrm [17–19], human homologues of the yeast SWI and SNF proteins that form the nucleosomal chromatin reorganizing SWI/SNF complex, and act as transcriptional activators. In nucleosomes, RB can also interact with the UBF transcription factor and can inhibit polymerase I [16]. RB can also repress polymerase III transcription, too [20]. RB can repress transcription by direct binding to promoters [21, 22]. In the case of E2F-, Sp1-, AP-1-, or p53-dependent transcriptional activation, a RB-GAL4 fusion protein represses transcriptional activation by this wide range of transcription factors, which includes factors typically associated with metabolic turn on. Finally RB can be a component of the basal transcriptional complex, where it exerts a negative regulatory influence [23–27]. The repressive activity depends on an interaction between the A and B pocket region domains and is relieved by phosphorylation by G1 cdk’s. The A region can specifically bind the TBP subunit of the TFIID factor, which begins assembly of general transcription factors at the TATA sequence. TBP participates in the initiation of transcrip-

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tion of all genes. The B region can specifically bind the TFIIB factor of the basal transcription complex. Hypophosphorylation of RB may thus have a more global regulatory function by regulating general levels of transcriptional activity, thereby determining if there is sufficient metabolic activity to sustain the ability of cells to transit the cell cycle. In the case of HL-60 myeloblastic leukemia cells, the importance of metabolic slow down for precipitating G0 arrest and differentiation has already been demonstrated by studies showing that withdrawal of a single essential amino acid can cause arrest and differentiation [28]. One anticipation of such suppositions is that in some cases it may be possible to witness the occurrence of hypophosphorylated RB slowing anabolism to render the cell unable to further transit the cell cycle regardless of what phase it is in. In this case, there should be a parallel between occurrence of hypophosphorylated RB and growth inhibition not necessarily restricted to G1/ 0 specific arrest. The HL-60 human myeloblastic leukemia cell line [29, 30] has been an archetype model for studies of cell differentiation and cell cycle control [31, review] for almost 20 years. The cells were derived from the peripheral blood of a patient with acute promyelocytic leukemia that was retrospectively reclassified as myeloblastic [32]. The cells proliferate avidly in culture and can be induced to undergo G0 arrest with either myeloid or monocytic differentiation. In contrast to other cell lines which might differentiate along only one lineage, HL-60 cells are an uncommitted precursor cell that can recapitulate in vitro the conversion of a proliferatively active immature cell to a differentiated proliferatively quiescent cell. DMSO or retinoic acid, for example, induce G0 arrest and myeloid differentiation, whereas sodium butyrate or 1,25-dihydroxy vitamin D3 induce monocytic differentiation. These inducers typically initiate a metabolic cascade which extends over a period corresponding to approximately two division cycles, ca. 48 h, resulting in onset of G0 arrest and differentiation. By 96 h growth is arrested and most of the cells express a differentiated phenotype. The period corresponding to two division cycles leading to onset of G0 arrest and differentiation segregates into two steps [33, 34]. ‘‘Early’’ events, corresponding to the duration of the first cycle, prime the cells to differentiate without lineage specificity. The ‘‘late’’ events, corresponding to the ensuing cycle anteceding differentiation or arrest, determine whether the lineage is myeloid or monocytic. Given the relatively detailed knowledge of these kinetics, it is possible to accurately assess the occurrence of hypophosphorylated RB with respect to cell growth and the cell cycle. MATERIALS AND METHODS Cells and culture conditions. HL-60 human promyelocytic leukemia cells were maintained in continuous exponential growth in

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RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum in a humidified atmosphere of 5% CO2 as previously described [12]. Experimental cultures for assaying G1/0 arrest and phenotypic conversion were initiated in 10-ml cultures of 0.2 1 106 cells/ml. DMSO or 1 M sodium butyrate was added to final concentrations of 1.25% or 1 mM, respectively. At indicated times cells were harvested for DNA staining and flow cytometric analysis of cell cycle distribution or for determining the percentage of cells capable of inducible oxidative metabolism, a functional marker of mature monocytic or myeloid cells, measured by ability to reduce nitroblue tetrazolium (NBT) to formazan as previously described [12]. Cells (0.5 1 106) were harvested, centrifuged to a cell pellet, and resuspended in (0.5 ml) hypotonic propidium iodide DNA staining solution [12, 13] or 0.2 1 106 cells in 0.2 mL NBT staining solution. Experimental cultures for Western analysis of RB protein changes were created by initiating proliferating cells in 30-ml cultures at an initial density of 0.2 1 106 cells/ml with the inducers. At indicated times aliquots of 1 to 10 1 106 cells were harvested and fixed in 90% methanol by resuspending the harvested cell pellet in 0.1 ml phosphate-buffered saline (PBS) over which 0.9 ml methanol at 0807C was layered and vortexed into suspension as described previously [12]. The fixed cells were stored at 0207C until used for DNA staining and fluorescence-activated cell sorting based on DNA content. Cell sorting. Fluorescence-activated cell sorting based on cellular DNA content was performed as previously described [13]. Fixed cells suspended in 90% methanol were washed in 0.5 ml 1:5 diluted PBS. The cells were resuspended in 0.5 ml 1:5 diluted PBS to which 50 ml RNase (3368 U/mg, 13 mg/ml, RASE A, Worthington Chemicals, Inc.) was added and incubated at 47C for 30 min. The cells were centrifuged to a pellet and resuspended in 0.05 mg/ml propidium iodide in phosphate-buffered saline (PI/PBS). The final cell density was approximately 10 1 106 cells/ml. The cells were analyzed with a dual laser multiparameter fluorescence-activated cell sorter (Coulter Electronics, Inc.) previously equilibrated to a running sheath fluid of 1:5 diluted PBS cooled to 47C. This PBS concentration obviated any salt extraction of proteins while still permitting droplet deflection. Cell sorting proceeded with an analysis rate of approximately 500 events/s. The 488-nm excitation was provided by a tunable argon ion laser operated at 200 mW (Coherent Laser, Inc.). Sorting was done with a three-droplet deflection envelope and coincidence rejection. Collection was into glass tubes containing chilled PI/PBS staining solution. Simultaneous sort fractions of G1 and S, G1 and G2 / M, or S and G2 / M were collected for each sample. Each of the sorted fractions were reanalyzed to verify purity. Sorted cell samples were stored as an aspirated pellet at 01007C until Western analysis. Unsorted cell samples were harvested and likewise stored until Western analysis. As described previously [13], Western analysis of cells prepared this way showed no loss of either the hyperphosphorylated or hypophosphorylated RB protein compared to freshly harvested samples. Untreated cells, which contain hyperphosphorylated RB protein, and inducer treated cells, which also contain hypophosphorylated RB protein, were used to confirm that there was no detectable loss due to the fixing and sorting protocol used. It should be noted that if the PBS sheath fluid used in sorting is not diluted as described, RB protein can be leached from the cells. Western analysis. Western analysis of RB protein expression was performed essentially as previously described [12, 13]. Cell pellets were resuspended in lysis buffer at the time of analysis. All lanes are loaded with lysate from an equal number of cells, ca. 106. The RB protein was resolved on a 6% PAGE running gel, transferred to a nitrocellulose membrane by electroblotting, and detected with a primary antibody (Zymed Laboratories, South San Francisco, CA) which recognized both the unphosphorylated and the phosphorylated protein, using an enhanced chemiluminescence developing kit (Amersham, Inc.) to create an image of the blot on X-ray film. The film

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FIG. 1. Cell number per milliliter (top) percentage of cells with G1 DNA (middle), and percentage of cells expressing inducible oxidative metabolism (bottom), a functional differentiation marker characteristic of mature myelomonocytic cells, are shown for cultures treated with 1 mM sodium butyrate (closed square) or 1.25% DMSO (closed triangle) as well as untreated control (open circle) cells. The horizontal axis shows time (hours) in culture. Determination of cell density and viability by hemacytometer counts, of %G1 DNA cells by propidium iodidestained nuclei analyzed by flow cytometry, and percentage of cells capable of inducible oxidative metabolism measured by intracellular reduction of nitroblue tetrazolium (NBT) were performed as previously described [12, 13]. There was no loss of viability (typically 95% throughout) incurred during culture detectable by trypan blue exclusion except when population decline occurred at late times. Data shown are typical of three repeats.

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FIG. 2. DNA histograms of (left to right) untreated control cells, 72 h DMSO-, and 72 h sodium butyrate-treated cells in the absence (top) or presence (bottom) of 1 mM vinblastine added during the last 10 h prior to harvesting. Control cells were cultured for 48 h to make their final cell density comparable to that of DMSO- or sodium butyrate-treated cells. The presence of cycling cells would be revealed by the accumulation of cells in G2/M by vinblastine. Control cells show this characteristic accumulation. Were it present, progress through the cell cycle in the sodium butyrate or DMSO growth-arrested populations would be detected by the accumulation of cells in the G2/M DNA peak where they would be blocked by the vinblastine. The effectiveness of this vinblastine G2/M block and lack of toxicity to interphase cell cycle transit has been shown previously [39]. The percentages of cells with G1 and G2 DNA (G1/G2) for control, DMSO-, and sodium butyrate-treated cells without versus with vinblastine are ca. 57/10 vs 32/30 (control), 88/4 vs 88/5 (DMSO), and 94/4 vs 94/3 (sodium butyrate). As shown by the decreasing %G1 and increasing %G2 of control cells after vinblastine treatment, 10 h is sufficient for G1 cells to reach G2. Thus in the case of DMSO-treated cultures, for example, the presence of cycling cells would cause the %G2 to increase during the 10 h of vinblastine treatment from 5% to over 12%, since the 7% of S phase cells plus any remaining cycling G1 cells would have reached G2. was scanned with a microprocessor-driven densitometer (Joyce-Loebl Ltd.) to derive densitometric traces of RB protein versus gel position. All scans were normalized to a scan axis of 20 mm.

RESULTS

HL-60 cells undergo population growth inhibition within ca. 1.5 population doublings when treated with 1 mM sodium butyrate and two doublings when treated with 1.25% DMSO. Figures 1a and 1b show the population growth and percentage of cells in G1/0 after initiation in culture with either 1 mM sodium butyrate or 1.25% DMSO as well as untreated controls. Figure 1c shows the percentage of cells capable of inducible oxidative metabolism, a functional marker for mature myelomonocytic cells. The saturation cell density of sodium butyrate treated cells is approximately 3 times the initial density of the starting exponentially proliferating population. This is associated with over 90% of the cells in G1/0. The saturation density of DMSOtreated cells is approximately 4 times the initial cell density. This is associated with approximately 80% of the cells in G1/0. Since population growth has reached saturation, both treated populations contain relatively small residual numbers of growth-arrested cells in S and G2. The residual non-G1 cells are not transiting the cell cycle. Figure 2 shows the DNA histograms for

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cells treated with sodium butyrate or DMSO for 72 h with 1006 M vinblastine added during the last 10 h. There is no accumulation of cells in G2/M, compared to parallel cultures without vinblastine, as would be expected if the cells were still cycling. This is consistent with the lack of increase in cell numbers. In contrast, proliferating control cells not treated with DMSO or sodium butyrate show an accumulation of cells in G2/M after 10 h of treatment with vinblastine. Unlike sodium butyrate- or DMSO-treated cells, control cells show unabated growth until a maximum density of approximately 10 times the initial density, with subsequent culture degeneration associated with nutritional exhaustion (data not shown). Consistent with this there was no significant G1/0 enrichment. Both sodium butyrate and DMSO thus induce growth arrest with a preponderance of cells, but not all, in G0; however, sodium butyrate inhibits growth more rapidly than DMSO. Hypophosphorylation of RB occurs in S and G2 as well as G1/0 for growth-inhibited cells induced by sodium butyrate or DMSO. Sodium butyrate causes hypophosphorylated RB to occur more rapidly in S and G2 than DMSO, which is a slower acting growth inhibitor. Figure 3A shows the densitometric scans from a Western analysis of RB protein in G1, S, and G2/M cells from cultures treated with sodium butyrate for 24, 48,

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FIG. 3. (A) Densitometric scans of autoradiographs from the Western blot analysis of RB protein expressed in (left to right) G1/0 DNA cells, S phase cells, and G2/M DNA cells isolated by fluorescence activated cell sorting on the basis of DNA content from (top to bottom) cells treated with sodium butyrate for 24, 48, and 72 h. Fluorescence-activated cell sorting of propidium iodide-stained cells and Western analysis of RB protein were performed as previously described [13]. Sorted cell fractions were reanalyzed to verify their purity as previously described [13]. The horizontal axis corresponds to 20 mm of the gel with the bottom of the gel oriented to the right. Each lane of the densitometrically scanned Western blot contains the total cell lysate of ca. 106 cells. The faster migrating hypophosphorylated RB protein appears as a distinct, narrower peak to the right of the broader, hyperphosphorylated RB protein peak. The approximate fractions of RB protein in the hypophosphorylated state for G1/S/G2 DNA cells after 24, 48, and 72 h of sodium butyrate are 0/0/0, 1.0/0.3/0.2, and 1.0/0.2/ 0.1 respectively. (B) Sort gates defining the G1/0, S, and G2/M fractions shown with the DNA histogram of a HL-60 cell population prior to sorting. The G1/0 gates are the left pair of vertical lines, and the G2/M gates are the right pair of vertical lines. The S phase gate (not shown) is between them. Sorted populations were reanalyzed to confirm their fidelity to these sort gates. The histogram is for whole cells and matches histograms derived from hypotonically derived nuclei, showing no evidence of endoreduplication in the HL-60 cells used. There are no cells with greater than 4n DNA as might be expected if some 4n DNA cells were in G1 (tetraploid) and proliferating, and treatment of cells with retinoic acid results only in a G1/0 DNA population, indicating no endoreduplication [12, 13, 39]. The HL-60 cells sorted by DNA content in this way should thus bear fidelity to G1/0, S, and G2/M cell cycle phases.

and 72 h (the x-axis of the scan corresponds to 20 mm on the gel with the bottom of the gel oriented to the right). The cell cycle phase specific subpopulations were derived by flow cytometric cell sorting on the basis of DNA content (Fig. 3B) as previously described [13]. The RB protein is 105 kDa, and phosphorylation inhibits its relative rate of migration in PAGE, allowing the faster migrating hypophosphorylated RB to be resolved as a distinct faster migrating species from the hyperphosphorylated RB protein, which migrates as a ca. 110-kDa protein. At 24 h all of the RB protein is still hyperphosphorylated. As shown previously [12, 13, 35, 36], in proliferating HL-60 cells all RB protein is hyperphosphorylated, and the extent of hyperphosphorylation increases as cells progress from the beginning to the end of the cell cycle (see also Fig. 5). At 48 h when growth is inhibited, RB has converted from the hyperto the hypophosphorylated form. In G1/0 cells, all of the RB protein is hypophosphorylated (see also Table 1). In the residual S phase cells of these growth inhib-

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ited populations, a significant fraction (approximately 0.3) of the RB protein is now also hypophosphorylated. This is also the case for residual G2 cells. At 72 h,

TABLE 1 Fraction of RB Protein Hypophosphorylated

Sodium butyrate 24 h 48 h 72 h DMSO 24 h 48 h 72 h

G1

S

G2

0 1.0 1.0

0 0.3 0.2

0 0.2 0.1

0 1.0 1.0

0 0 0.1

0.1 0.3 0.6

Note. Fraction of RB protein hypophosphorylated for cells in the indicated cell cycle phases after treated with sodium butyrate or DMSO for the indicated times.

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untreated control cells, there is no hypophosphorylated RB in cells of any cell cycle phase (Fig. 5 shows the Western blot of RB protein for both DMSO- and sodium butyrate-treated cells). After 48 h of DMSO treatment, cell growth is arrested. At this time all of the RB protein in G1/0 cells is hypophosphorylated as in the case of sodium butyrate. In S phase, there is no clearly detectable hypophosphorylated RB protein, unlike the case of sodium butyrate. In G2 cells, a significant fraction (approximately 0.3) of the RB protein is hypophosphorylated. At 72 h all of the RB protein in G1/0 cells remains hypophosphorylated and S and G2 cells both now also have hypophosphorylated RB. The occurrence of hypophosphorylated RB in both S and G2 thus lags slightly behind the case of sodium butyrate. DMSOtreated cells also did not growth arrest as quickly as sodium butyrate-treated cells and reached a higher plateau cell density than sodium butyrate-treated cells. DISCUSSION FIG. 4. Densitometric scans as in Fig. 3 of autoradiographs from the Western blot analysis of RB protein expressed in (left to right) G1/0 DNA cells, S phase cells, and G2/M DNA cells isolated by fluorescence-activated cell sorting on the basis of DNA content from (top to bottom) cells treated with DMSO for 24, 48, and 72 h. Fluorescence activated cell sorting of propidium iodide-stained cells and Western analysis of RB protein were performed the same as for Fig. 3. The approximate fractions of RB protein in the hypophosphorylated state for G1/S/G2 DNA cells after 24, 48, and 72 h of DMSO are 0/0/0.1, 1.0/0/0.3, and 1.0/0.1/0.6, respectively.

when there is no further growth in cell numbers or enrichment in G1/0 cells, a similar pattern of RB hypophosphorylation with respect to G1/0, S, and G2 is again observed. All of the RB protein of G1/0 cells is hypophosphorylated. In the residual S and G2 cells, which still comprise approximately 10% of the population, a clearly observable fraction of the RB protein is hypophosphorylated. Thus hypophosphorylated RB is most extensively induced in G1/0-arrested cells, but is also induced to a lesser extent in S and G2 cells which are arrested in their progress through the cell cycle. The extent of induced RB hypophosphorylation with respect to cell cycle phase roughly parallels where the most prominent arrest is, that is, G1 and to a lesser extent S and G2. Sodium butyrate thus appears to cause RB hypophosphorylation most strongly in G1 cells and to a lesser extent in S and G2 cells, resulting in the most prominent cell cycle phase arrest in G1/0 and to a lesser extent in S and G2. Another inducer of HL-60 growth arrest, DMSO, causes similar effects on RB hypophosphorylation, indicating that these effects are not unique to sodium butyrate. Figure 4 shows the densitometric scans from a Western analysis of RB protein in G1, S, and G2/M cells sorted from cultures treated with DMSO for 24, 48, and 72 h. At 24 h when cell growth still matches

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The data show that hypophosphorylation of RB occurs not just in G1/0 but also in S and G2 where it can also arrest cell cycle progression. One simple rationalization of these observations is that sodium butyrate or DMSO induce RB hypophosphorylation most effectively in G1, causing the most prominent arrest there, but RB hypophosphorylation is also induced at a lesser rate in S and G2 ultimately also causing cells to be arrested there, too, although not as quickly as in G1/0. Consistent with this, sodium butyrate induces the occurrence of hypophosphorylated RB in all cell cycle phases earlier than DMSO and also induces growth arrest of the population faster than DMSO. Inherent in this simple rationalization is the implicit assumption that in collected S or G2/M cells the fraction of RB protein which is hypophosphorylated measured by Western analysis approximates the fraction of RB protein which is hypophosphorylated per average cell in the S or G2/M subpopulations. This is the simplest model. An alternative model is that the subpopulation is heterogeneous with only a fraction of cells bearing hypophosphorylated RB protein. The origin of this heterogeneity might be attributable to differences arising with progression within these specific cell cycle phases or with differentiation state. This ambiguity is necessitated by Western analysis which can only analyze cell populations and not individual cells. The present data do not allow any rigorous distinction between these alternative models. However, there are no compelling data indicating that there are arbitrary sources of heterogeneity giving rise to cells with and without hypophosphorylated RB in the individually sorted phases. The minimal model providing a rationalization of the data has thus been adopted above.

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FIG. 5. (A) Western blot of RB protein in (left to right in each panel) G1/0, S, and G2/M cells, isolated by fluorescence activated cell sorting according to DNA content, for cells treated with 1.25% DMSO for (left to right panels) 24, 48, and 72 h. The lower molecular weight apparent degradation product [39] in the 72-h G2/M lane appears below the hypophosphorylated RB occasionally, but not in all repeats. (B) Western blot of RB protein in (left to right in each panel) G1/0, S, and G2/M cells, isolated by fluorescence activated cell sorting according to DNA content, for cells treated with 1 mM sodium butyrate for (left to right panels) 24, 48, and 72 h.

These data conform to the anticipations of the paradigm suggested earlier where RB has a role in regulating general levels of cellular anabolism. Thus an RBdependent reduction of general transcriptional activity may result in a metabolic slow down rendering HL60 cells unable to continue transit through the cycle. Occurrence of hypophosphorylated RB in cell cycle phases other than G1/0 might thus lead to arrest in those phases, too. One can still only speculate why dephosphorylation of RB is most thorough in G1 cells, where arrest is most prominent. Several factors in combination may conspire toward this end. One is that in proliferating HL-60 cells RB is the least phosphorylated in G1 compared to S and G2 [13]. The amount of RB protein per cell as well as its extent of phosphorylation increases with progression through the cell cycle [37, 38]. There is thus also less RB protein to convert in G1 compared to S and G2. These may contribute to the conversion of all RB protein to the hypophosphorylated state first in G1 compared to S and G2. A simple rationalization of the present data is thus that general levels of cellular anabolism depend on RB acting at the transcriptional level, and the induced conversion of RB to the hypophosphorylated state contributes to metabolic slow down, thereby inhibiting continued progression through the cell cycle. Because RB in G1 cells is least phosphorylated and there is less RB protein per cell compared to subsequent cell cycle phases, conversion of the RB protein to the hypophosphorylated state is most rapid in G1 cells, causing the most prominent arrest there. Contributing to this is the long known fact that, compared to other cell cycle phases, G1 cells typically have the most pronounced sensitivity to inhibition of protein synthesis. Residual cells escaping G1 arrest incur hypophosphorylated RB in subsequent cell cycle phases and finally arrest in later cell cycle phases, S and G2. Consistent with this paradigm, it is of interest to note anecdotally that inhibiting src-kinases in HL-60 cells results in a transient hypophosphorylation of RB associated with a transient G1 arrest [36].

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Received October 7, 1997 Revised version received January 13, 1998

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