CDK4/6 Inhibition With Lerociclib is a Potential Therapeutic Strategy for the Treatment of Pediatric Sarcomas.

BACKGROUND: Sarcomas are a heterogenous collection of bone and soft tissue tumors. The heterogeneity of these tumors makes it difficult to standardize treatment. CDK 4/6 inhibitors are a family of targeted agents which limit cell cycle progression and have been shown to be upregulated in sarcomas. In the current preclinical study, we evaluated the effects of lerociclib, a CDK4/6 inhibitor, on pediatric sarcomas in vitro and in 3D bioprinted tumors. METHODS: The effects of lerociclib on viability, proliferation, cell cycle, motility, and stemness were assessed in established sarcoma cell lines, U-2 OS and MG-63, as well as sarcoma patient-derived xenografts (PDXs). 3D printed biotumors of each of the U-2 OS, MG-63, and COA79 cells were utilized to study the effects of lerociclib on tumor growth ex vivo. RESULTS: CDK 4/6, as well as the intermediaries retinoblastoma protein (Rb) and phosphorylated Rb were identified as targets in the four sarcoma cell lines. Lerociclib treatment induced cell cycle arrest, decreased proliferation, motility, and stemness of sarcoma cells. Treatment with lerociclib decreased sarcoma cell viability in both traditional 2D culture as well as 3D bioprinted microtumors. CONCLUSIONS: Inhibition of CDK 4/6 activity with lerociclib was efficacious in traditional 2D sarcoma cell culture as well as in 3D bioprints. Lerociclib holds promise and warrants further investigation as a novel therapeutic strategy for management of these heterogenous groups of tumors.

Dual-site regulation of MDM2 E3-ubiquitin ligase activity.

The control of p53 ubiquitination by MDM2 provides a model system to define how an E3-ligase functions on a conformationally flexible substrate. The mechanism of MDM2-mediated ubiquitination of p53 has been analyzed by deconstructing, in vitro, the MDM2-dependent ubiquitination reaction. Surprisingly, ligands binding to the hydrophobic cleft of MDM2 do not inhibit its E3-ligase function. However, peptides from within the DNA binding domain of p53 that bind the acid domain of MDM2 inhibit ubiquitination of p53, localizing a motif that harbors a key ubiquitination signal. The binding of ligands to the N-terminal hydrophobic cleft of MDM2 reactivates, in vitro and in vivo, MDM2-catalyzed ubiquitination of p53F19A, a mutant p53 normally refractory to MDM2-catalyzed ubiquitination. We propose a model in which the interaction between the p53-BOX-I domain and the N terminus of MDM2 promotes conformational changes in MDM2 that stabilize acid-domain interactions with a ubiquitination signal in the DNA binding domain of the p53 tetramer.

HES1 cooperates with pRb to activate RUNX2-dependent transcription.

UNLABELLED: The retinoblastoma protein, pRb, can activate the transcription factor RUNX2, an essential regulator of osteogenic differentiation, but the mechanism of this activation is unknown. Here we studied the interaction of pRb and RUNX2 with HES1, previously reported to augment RUNX2 activity. PRb can act to promote RUNX2/HES1 association with concomitant promoter occupancy and transcriptional activation in bone cells. INTRODUCTION: RUNX2 (also known as OSF2/CBFA1) is a transcription factor required for osteoblast differentiation and bone formation. We have reported that RUNX2 can associate with the retinoblastoma protein pRb, a common tumor suppressor in bone, and the resultant complex can bind and activate transcription from bone-specific promoters. This activity of the pRb/RUNX2 complex may thus link differentiation control with tumor suppressor activity. However, the mechanism through which pRb can activate RUNX2 is unknown. HES1 is a reported co-activator of RUNX2 that shares a binding site on RUNX2 with pRb. Thus, we have tested the cooperativity among these factors in activating transcription from bone specific promoters. MATERIALS AND METHODS: Coimmunoprecipitation, chromatin immunoprecipitation, and EMSA experiments were used to study the interaction of RUNX2, HES1, and pRb in cell lysates and on DNA. Transcriptional reporter assays were used to analyze the activity of RUNX2 in the presence and absence of HES1 and pRb. RESULTS: We showed that pRb can associate with HES1, a previously described RUNX2 interactor that can itself augment RUNX2-dependent transcription. The association of HES1 with RUNX2 is augmented by pRb. Furthermore, both pRb and HES1 increase the amount of RUNX2 bound to promoter sites in vivo, pRb and HES1 synergistically activate a RUNX2-dependent reporter gene, and depletion of HES1 reduces RUNX2/pRb activity. CONCLUSIONS: These data indicate that pRb acts as a RUNX2 co-activator at least in part by recruiting HES1 into the pRb/RUNX2 complex and further elucidate a novel role for pRb as a transcriptional co-activator in osteogenesis.

Effect of exogenous p16ink4a and hRb1 genes on cell cycle regulation of osteosarcoma cell.

To study the effect on regulation of cell cycle of osteosarcoma cell line MG63 tranceduced with exogenous p16ink4a and hRb1 genes, pIRES-p16ink4a-hRb1, pIRES-p16ink4a and pIRES-hRb1 plasmids were constructed by gene recombination technology. The recombinant plasmid was transferred into osteosarcoma cell line MG63 by metafectene, and the resistant clones were selected by G418 selective medium. mRNA and protein expression of osteosarcoma cell line were assayed by RT-PCR and Western-Blot respectively. Cell cycle and apoptosis were analyzed by subG1 flow cytometric. Cell proliferation was tested by MTT. In the genome of these transfected target cells, the expression of p16ink4a and hRb1 mRNA and protein were detected respectively in vitro. It was demonstrated with subG1 flow cytometric analysis and MTT method that p16ink4a and hRb1 genes cooperation more significantly inhibited cell growth and induced a more marked G1 arrest and apoptosis than p16ink4a/hRb1 alone (P < 0.01). Coexpression of exogenous p16ink4a with hRb1 broke the regulatory feedback loop of p16ink4a-cyclinD1 /CDK-hRb1 and played a more significant role in inhibiting cell growth as well as inducing cell apoptosis than p16ink4a or hRb1 did alone in vitro.

Altered expression of cell cycle regulators Cyclin D1, p14, p16, CDK4 and Rb in nodular melanomas.

Cell cycle regulating proteins are important in tumour development. To investigate whether alterations in Cyclin D1, p14, CDK4 and Rb are associated with tumour cell proliferation, tumour progression and patient survival in malignant melanoma, we examined 202 vertical growth phase tumours and 68 corresponding metastases for expression of Cyclin D1, p14, CDK4 and Rb, and compared the results with Ki-67 expression, p16 and p53 expression, clinico-pathological variables, and survival data. Nuclear staining of Cyclin D1 was strong in 35% of cases, and correlated with high levels of Rb (p=0.05), but not with survival or other markers tested. Strong staining of p14 was found in 63% of nodular melanomas and was associated with strong p53 expression (p=0.014), and with high levels of CDK4 (p<0.0001). Low p14 expression was associated with increased tumour thickness (p=0.008) and increasing level of invasion (p=0.020). Strong nuclear staining for CDK4 was found in 81% of cases and was associated with tumour thickness below the median value of 3.7 mm and improved survival (log-rank test, p=0.024). Further, 56% of the tumours showed strong nuclear staining for Rb, and these cases were significantly associated with absent/low levels of p16 staining (p=0.030), high levels of p14 (p=0.010), as well as high Ki-67 expression (p=0.005). Our results seem to confirm that the p16-Rb pathway plays an important role in tumour progression and prognosis in vertical growth phase melanomas, whereas alterations in the p14-p53 pathway might be less important.

A model for restriction point control of the mammalian cell cycle.

Inhibition of protein synthesis by cycloheximide blocks subsequent division of a mammalian cell, but only if the cell is exposed to the drug before the "restriction point" (i.e. within the first several hours after birth). If exposed to cycloheximide after the restriction point, a cell proceeds with DNA synthesis, mitosis and cell division and halts in the next cell cycle. If cycloheximide is later removed from the culture medium, treated cells will return to the division cycle, showing a complex pattern of division times post-treatment, as first measured by Zetterberg and colleagues. We simulate these physiological responses of mammalian cells to transient inhibition of growth, using a set of nonlinear differential equations based on a realistic model of the molecular events underlying progression through the cell cycle. The model relies on our earlier work on the regulation of cyclin-dependent protein kinases during the cell division cycle of yeast. The yeast model is supplemented with equations describing the effects of retinoblastoma protein on cell growth and the synthesis of cyclins A and E, and with a primitive representation of the signaling pathway that controls synthesis of cyclin D.

The dominant-negative effect of p53 mutants and p21 induction in tetraploid G1 arrest depends on the type of p53 mutation and the nature of the stimulus.

Many p53 mutant proteins possess a dominant-negative activity that is under the control of several factors, namely p53 mutations and the cell type. The goals of our study were to determine the following: (1) the dominant-negative effect of different p53 mutations in response to mitotic spindle inhibitors, and (2) if this dominant-negative activity is dependent on the nature of the stimulus. We therefore examined the cellular response of the near-diploid LoVo colon carcinoma cell line possessing two wild-type TP53 alleles and three other clones transfected with different TP53 mutants (p53-273H, p53-175H, and p53-143A) to treatments with different mitotic spindle inhibitors. Flow cytometric studies and analysis of retinoblastoma protein (pRb) dephosphorylation and 5-bromo-2'-deoxyuridine incorporation by immunocytochemistry revealed a tetraploid G1 arrest of the wild-type LoVo clone and the p53-273H mutant clone after exposure to mitotic spindle inhibitors, preventing tetraploid cells from entering into an additional S phase. On the other hand, the p53-175H and p53-143A mutant clones re-enter S phase with no apparent arrest. Therefore, our results confirm that p53 mutant dominant-negative activity and the tetraploid G1 arrest in response to mitotic spindle inhibitor treatment depend on the type of p53 mutation, involve p21 induction, and require pRb dephosphorylation. Moreover, when we compare our results with those obtained by other investigators after ionizing radiation exposure using the same cell lines, we identify the nature of the stimulus as a new factor that determines the dominant-negative effect of p53 mutants.

Yin Yang 1 is a lipopolysaccharide-inducible activator of the murine 3' Igh enhancer, hs3.

The 3' Igh enhancers, DNase I hypersensitive site (hs) 3B and/or hs4, are required for germline transcription, and hence, class switch recombination for multiple isotypes. A number of hs3-binding transcription factors have been identified by EMSA, including octamer and NF-kappa B family members, and Pax5. We have found that the binding of the transcription factor, Yin Yang 1 (YY1), to hs3 and to the mu E1 site of the intronic enhancer, E mu, is induced in primary splenic B cells after approximately 48 h in response to LPS and other activators of class switch recombination. Transient transfection experiments in B cell lines indicate that YY1 is an activator of hs3. Interestingly, levels of YY1 expression are unchanged in resting and LPS-stimulated B cells. Mixing experiments followed by EMSA showed that a protein present in resting B cells prevented binding of YY1 to DNA. We found that recombinant retinoblastoma protein (Rb) inhibited binding of YY1 to hs3 in a dose-dependent manner, and we have identified complexes of endogenous YY1 with the Rb in resting B cells, but not in LPS-stimulated B cells. A difference in Rb phosphorylation state was also confirmed between resting (G(0)) B cells and LPS-stimulated B cells. These observations suggest that the interaction of YY1 with hypophosphorylated Rb in resting B cells prevents interaction of YY1 with DNA. After stimulation with class-switching activators, such as LPS, Rb becomes hyperphosphorylated and YY1 is released and can then bind to the hs3 enhancer and E mu.

Distinct mechanisms for regulating the tumor suppressor and antiapoptotic functions of Rb.

The retinoblastoma protein, Rb, suppresses tumorigenesis by inhibiting cell proliferation and promoting senescence and differentiation. Paradoxically, Rb also inhibits apoptosis, which would seem to oppose its tumor suppressor function. Further, most human cancer cells inactivate Rb by hyperphosphorylation and demonstrate increased proliferative capacity but not high levels of apoptosis. As a potential explanation for these findings, we show here that the tumor suppressor and antiapoptotic functions of Rb are regulated by distinct phosphorylation events. Phosphorylation of sites in the C terminus occurs efficiently every cell cycle and regulates proliferation. Phosphorylation of Ser567 is inefficient and does not occur during the normal cell cycle. However, high cyclin-dependent kinase activity promotes phosphorylation of Ser567 by inducing an intramolecular interaction that leads to release of E2F, degradation of Rb, and susceptibility to apoptosis. Thus, phosphorylation of Ser567 may limit excessive proliferation by triggering cell death under hyperproliferative conditions. These findings suggest that the antiproliferative and antiapoptotic activities of Rb may represent complementary functions that work in concert to maintain the proliferation rate of cells within certain limits. As a survival strategy, some cancer cells may exploit this dual role of Rb by phosphorylating sites that regulate tumor suppression but avoiding phosphorylation of Ser567 and consequent apoptotic stimulus.

E2F activity is essential for survival of Myc-overexpressing human cancer cells.

Effective cell cycle completion requires both Myc and E2F activities. However, whether these two activities interact to regulate cell survival remains to be tested. Here we have analysed survival of inducible c-Myc-overexpressing cell lines derived from U2OS human osteosarcoma cells, which carry wild-type pRb and p53 and are deficient for p16 and ARF expression. Induced U2OS-Myc cells neither underwent apoptosis spontaneously nor upon reconstitution of the ARF-p53 axis and/or serum-starvation. However, they died massively when concomitantly exposed to inhibitors of E2F activity, including a constitutively active pRb (RbDeltacdk) mutant, p16, a stable p27 (p27T187A) mutant, a dominant-negative (dn) CDK2, or dnDP-1. Similar apoptotic effect was observed upon down-modulation of endogenous E2Fs through overexpression of E2F binding site oligonucleotides in U2OS-Myc cells, upon expression of RbDeltacdk or dnDP-1 in the Myc-amplified HL-60 (ARF-; p53-) human leukemia cells, and upon co-transfection of Myc and RbDeltacdk in SAOS-2 (ARF+; p53-) human osteosarcoma cells but not in human primary fibroblasts. Consistent with these results, a dnp53 mutant did not abrogate the Myc-induced apoptotic phenotype, which instead strictly depended on caspase-3-like proteases and on Myc transcriptional activity. Our data indicate that in contrast to normal cells, Myc-overexpressing human cancer cells need E2F activity for their survival, regardless of their ARF and p53 status, a notion that may have important implications for antineoplastic treatment strategies.

N-myc promotes survival and induces S-phase entry of postmitotic sympathetic neurons.

In most postmitotic neurons, expression or activation of proteins that stimulate cell cycle progression or DNA replication results in apoptosis. One potential exception to this generalization is neuroblastoma (NB), a tumor derived from the sympathoadrenal lineage. NBs often express high levels of N-myc, a proto-oncogene that can potently activate key components of the cell cycle machinery. Here, we show that in postmitotic sympathetic neurons, N-myc can induce S-phase entry while protecting neurons from death caused by aberrant cell cycle reentry. Specifically, these experiments demonstrate that expression of N-myc at levels similar to those in NBs caused sympathetic neurons to reenter S-phase, as monitored by 5-bromo-2-deoxyuridine incorporation and expression of cell cycle regulatory proteins, and rescued them from apoptosis induced by withdrawal of their obligate survival factor, nerve growth factor. The N-myc-induced cell cycle entry, but not enhanced survival, was inhibited by coexpression of a constitutively hypophosphorylated form of the retinoblastoma tumor suppressor protein, suggesting that these two effects of N-myc are mediated by separate pathways. In contrast, N-myc did not cause S-phase entry in postmitotic cortical neurons. Thus, N-myc both selectively causes sympathetic neurons to reenter the cell cycle and protects them from apoptosis, potentially contributing to their transformation to NBs.

Acetylation control of the retinoblastoma tumour-suppressor protein.

The retinoblastoma tumour-suppressor protein (pRb) and p300/CBP co-activator proteins are important for control of proliferation and in tumour cells these are sequestered by viral oncoproteins such as E1A. pRb is involved in negatively regulating growth, and p300/CBP proteins have histone acetyltransferase (HAT) activity, which influences gene expression. Although it is known that phosphorylation by G1 cyclin-dependent kinases (CDKs) regulates pRb activity, the nature and role of other post-translational modifications is not understood. Here we identify acetylation as a new type of modification and level of control in pRb function. Adenovirus E1A, which binds p300/CBP through an amino-terminal transformation-sensitive domain, stimulates the acetylation of pRb by recruiting p300 and pRb into a multimeric-protein complex. Furthermore, pRb acetylation is under cell-cycle control, and acetylation hinders the phosphorylation of pRb by cyclin-dependent kinases. pRb binds more strongly when acetylated to the MDM2 oncoprotein, which indicates that acetylation may regulate protein-protein interactions in the pRb pathway. The acetylation of pRb defines a new level of cell-cycle control mediated by HAT. Furthermore, our results establish a relationship between p300, pRb and acetylation in which E1A acts to recruit and target a cellular HAT activity to pRb.

Resistance to fulminant hepatitis and carcinogenesis conferred by overexpression of retinoblastoma protein in mouse liver.

Previously, retinoblastoma (Rb) transgenic mice were produced under the control of the Rb gene promoter and showed dwarf characteristics. Here, we created transgenic mice, in which the human Rb gene was controlled by the hepatocyte nuclear factor-1 gene promoter/enhancer and was expressed primarily in the liver. The liver of these novel transgenic mice was normally developed. Intriguingly, these mice showed resistance to fulminant hepatitis induced by anti-Fas antibody as well as resistance to chemical carcinogenesis in the liver. These results show that the Rb protein acts as an anti-apoptotic and anti-oncogenic agent in vivo. Our novel construct may be useful as a gene cassette in gene therapy for prevention of fulminant hepatitis and hepatoma.

Role of retinoblastoma tumor suppressor protein in DNA damage response.

Growth arrest induced by DNA damage in mammalian cells requires the function of the retinoblastoma tumor suppressor protein (RB). RB-deficient cells cannot undergo G1, mid-S or G2 arrest following DNA damage, although they can activate the G2 checkpoint, which is reversible. RB-deficient cells are also hypersensitive to DNA damage-induced apoptosis. Induction of apoptosis in RB wild-type cells is associated with the loss of RB protein through cleavage by caspase. Two substitution mutations in exon 25 of the Rb gene have been created in the mouse germline to generate the Rb-MI allele that codes for a caspase-resistant RB protein. The RB-MI protein desensitizes cells to apoptosis. Taken together, these results suggest that RB plays a critical role in determining the cell fate following DNA damage. Growth arrest is dependent on RB and apoptosis is activated following RB degradation.

Retinoblastoma protein in microphthalmic mice.

A microphthalmic strain of mice was used to study immunoresponse of the retinoblastoma protein. Comparing wild-type, heterozygote and homozygote microphthalmic eyes, we found an increasing labelling of phosphorylated retinoblastoma protein (pRb) in the retinal pigment epithelium. Additionally, microphthalmic eyes expressed pRb in the neuroepithelium. Especially rosettes were strongly labelled.

Selective inhibition of human lung cancer cell growth by peptides derived from retinoblastoma protein.

Peptides containing retinoblastoma protein (RB) fragment 649-654 (LFYKKV) were tested for their ability to block the proliferation of RB-negative and RB-positive human non-small cell lung cancer (NSCLC) cells. These peptides potently restrained the growth of both types of tumor cells, as measured by metabolic (MTT) and cellular viability (trypan blue exclusion) assays. As such, and remarkably, the peptides were able to overcome the resistance of RB-positive cells usually observed with RB gene or protein replacement therapy. Compared to the overall performance of conventional chemotherapy tested in parallel, the peptides were more cytotoxic against RB-negative neoplastic cells and equipotent toward RB-positive tumor cells, yet less toxic toward normal human cells. Thus, these new molecules hold great promise to evolve into an efficient therapy for human lung cancer, a common malignancy still defying treatment and holding a poor prognosis, as well as for other human neoplasias.

Activation of telomerase rna gene promoter activity by NF-Y, Sp1, and the retinoblastoma protein and repression by Sp3.

Expression of the human telomerase RNA component gene, hTERC is essential for telomerase activity. The hTERC gene is expressed during embryogenesis and then downregulated during normal development, leaving most adult somatic cells devoid of hTERC expression. During oncogenesis, however, hTERC is re-expressed consequently contributing to the unrestricted proliferative capacity of many human cancers. Thus the identification of the molecular basis for the regulation of the telomerase RNA component gene in normal cells and its deregulation in cancer cells is of immediate interest. We have previously cloned the hTERC promoter and in this study have identified several transcription factors that modulate the expression of hTERC. We demonstrate that NF-Y binding to the CCAAT region of the hTERC promoter is essential for promoter activity. Sp1 and the retinoblastoma protein (pRb) are activators of the hTERC promoter and Sp3 is a potent repressor. These factors appear to act in a species-specific manner. Whereas Sp1 and Sp3 act on the human, bovine, and mouse TERC promoters, pRb activates only the human and bovine promoter, and NF-Y is only essential for the human TERC gene.

Differential induction of the androgen receptor transcriptional activity by selective androgen receptor coactivators.

Several new androgen receptor (AR) cofactors, associated to the ligand binding domain of AR, have been identified by our group and named AR associated protein (ARA)70, ARA55, and ARA54. Our previous reports have suggested that the cofactor ARA70 can confer the androgenic effect from 17 beta-estradiol (E2) and antiandrogen to AR. It is of interest for us to compare and determine if the specificity of sex hormones and antiandrogens could be modulated by different coactivators. Our results indicate that ARA70 is the best coactivator to confer the androgenic activity on E2. Only ARA70 and ARA55 could increase significantly the androgenic activity of hydroxyflutamide, a widely used antiandrogen for the treatment of prostate cancer. Furthermore, as compared to the relative specificity of these coactivators to AR in the prostate cancer DU145 cells, our results suggest that ARA70 has a relatively higher specificity. Together, our data suggest that the specificity of sex hormones and antiandrogens can be modulated by some selective AR coactivators. These findings may not only help us to better understand the specificity of the sex hormones and antiandrogens, but also to facilitate the development of better antiandrogens or androgens to fight the androgen-related diseases, such as prostate cancer.

Functional interplay between p53 and E2F through co-activator p300.

Both E2F and p53 are sequence specific transcription factors that regulate early cell cycle progression. The pathway of control mediated through E2F governs the transition from G1 into S phase whereas p53 in response to genotoxic stress can facilitate cell cycle arrest or apoptosis. The mechanisms which influence the outcome of p53 induction are not clear, although transcription of the p53 target gene, encoding the cdk-inhibitor p21(Waf1/Cip1), correlates with p53-mediated cell cycle arrest. Here using a combination of biochemical and functional assays we identify p300 as a co-activator required for p53-dependent transcriptional activation of Waf1/Cip1. Furthermore, we show that the cdk-inhibitor p21(Waf1/Cip1) autoregulates in a positive fashion transcription through modulating the activity of the p53/p300 complex, whilst negatively regulating the activity of E2F by preventing cdk-dependent phosphorylation of pRb. Consistent with a role for p21(Waf1/Cip1) in the autoregulation of p53-dependent transcription, p300 augments the ability of p53 to cause G1 arrest and, conversely, cells undergoing p53-dependent apoptosis are rescued by p300. Thus, our data suggest that the ability of p300 to interact with p53 influences the physiological consequence of p53 activation. From previous studies it is known that cells expressing aberrant levels of E2F-1 can undergo p53-dependent apoptosis. In addition, we find that E2F-1 can cause apoptosis in p53-/- tumour cells and further p300, which also functions as a co-activator for the E2F/DP heterodimer, enhances the apoptotic activity of E2F-1. In conditions where E2F-1 and p53 co-operate in apoptosis E2F-1 can effectively compete for p300, causing a reduction in p53-dependent transcription. Thus, a functional interaction between p300 and either p53 or E2F-1 has a profound impact on early cell cycle progression, specifically in regulating the contrasting outcomes of cell cycle arrest and apoptosis. These results suggest a critical role for p300 in integrating and co-ordinating the functional interplay between the pathways of growth control mediated by E2F and p53.

p53 and pRb prevent rereplication in response to microtubule inhibitors by mediating a reversible G1 arrest.

Cell cycle checkpoints are safeguards that ensure the initiation of downstream events only after completion of upstream processes. The tumor suppressors p53 and pRb prevent initiation of a second round of replication in response to spindle inhibitors, but it has yet to be proven that this is a mitotic checkpoint response. We show that asynchronous human fibroblasts arrest in G1 with 4 N DNA content after nocodazole treatment, whereas isogenic p53- and pRb-deficient fibroblasts rereplicate. Importantly, nocodazole elicits a reversible arrest in G0-G1 synchronized normal human fibroblasts but not in isogenic p53-deficient derivatives. Furthermore, the G1 cyclin-dependent kinase inhibitors p21 and p16 also play critical roles in limiting rereplication. Hence, p53 and pRb are required during G1 to prevent entry into a replicative cycle and appear to provide a connection between the structural integrity of the microtubules and the cell cycle machinery in interphase cells.