Opposing early and late effects of insulin-like growth factor I on differentiation and the cell cycle regulatory retinoblastoma protein in skeletal myoblasts.

The mechanisms by which insulin-like growth factors (IGFs) can be both mitogenic and differentiation-promoting in skeletal myoblasts are unclear because these two processes are believed to be mutually exclusive in this tissue. The phosphorylation state of the ubiquitous nuclear retinoblastoma protein (Rb) plays an important role in determining whether myoblasts proliferate or differentiate: Phosphorylated Rb promotes mitogenesis, whereas un- (or hypo-) phosphorylated Rb promotes cell cycle exit and differentiation. We hypothesized that IGFs might affect the fate of myoblasts by regulating the phosphorylation of Rb. Although long-term IGF treatment is known to stimulate differentiation, we find that IGFs act initially to inhibit differentiation and are exclusively mitogenic. These early effects of IGFs are associated with maintenance of Rb phosphorylation typical of proliferating cells; upregulation of the gene expression of cyclin-dependent kinase 4 and cyclin D1, components of a holoenzyme that plays a principal role in mediating Rb phosphorylation; and marked inhibition of the gene expression of myogenin, a member of the MyoD family of skeletal muscle-specific transcription factors that is essential in muscle differentiation. We also find that IGF-induced inhibition of differentiation occurs through a process that is independent of its mitogenic effects. We demonstrate, thus, that IGFs regulate Rb phosphorylation and cyclin D1 and cyclin-dependent kinase 4 gene expression; together with their biphasic effects on myogenin expression, these results suggest a mechanism by which IGFs are initially mitogenic and subsequently differentiation-promoting in skeletal muscle.

Identification of a 95-kDa WEE1-like tyrosine kinase in HeLa cells.

Human WEE1 (WEE1Hu) was cloned on the basis of its ability to rescue wee1+ mutants in fission yeast [Igarashi, M., Nagata, A., Jinno, S., Suto, K. & Okayama, H. (1991) Nature (London) 353, 80-83]. Biochemical studies carried out in vitro with recombinant protein demonstrated that WEE1Hu encodes a tyrosine kinase of approximately 49 kDa that phosphorylates p34cdc2 on Tyr-15 [Parker, L. L. & Piwnica-Worms, H. (1992) Science 257, 1955-1957]. To study the regulation of WEE1Hu in human cells, two polyclonal antibodies to bacterially produced p49WEE1Hu were generated. In addition, a peptide antibody generated against amino acids 361-388 of p49WEE1Hu was also used. Unexpectantly, these antibodies recognized a protein with an apparent molecular mass of 95 kDa in HeLa cells, rather than one of 49 kDa. Immunoprecipitates of p95 phosphorylated p34cdc2 on Tyr-15, indicating that p95 is functionally related to p49WEEIHu, and mapping studies demonstrated that p95 is structurally related to p49WEE1Hu. In addition, the substrate specificity of p95 was more similar to that of fission yeast p107wee1 than to that of human p49WEE1. Finally, the kinase activity of p95 toward p34cdc2/cyclin B was severely impaired during mitosis. Taken together, these results indicate that the original WEE1Hu clone isolated in genetic screens encodes only the catalytic domain of human WEE1 and that the authentic human WEE1 protein has an apparent molecular mass of approximately 95 kDa.

Induction of a retinoblastoma phosphatase activity by anticancer drugs accompanies p53-independent G1 arrest and apoptosis.

DNA-damaging agents induce accumulation of the tumor suppressor and G1 checkpoint protein p53, leading cells to either growth arrest in G1 or apoptosis (programmed cell death). The p53-dependent G1 arrest involves induction of p21 (also called WAF1/CIP1/SDI1), which prevents cyclin kinase-mediated phosphorylation of retinoblastoma protein (RB). Recent studies suggest a p53-independent G1 checkpoint as well; however, little is known about its molecular mechanisms. We report that induction of a protein-serine/threonine phosphatase activity by DNA damage signals is at least one of the mechanisms responsible for p53-independent, RB-mediated G1 arrest and consequent apoptosis. When two p53-null human leukemic cell lines (HL-60 and U-937) were treated with a variety of anticancer agents, RB became hypophosphorylated, accompanied with G1 arrest. This was followed immediately (in less than 30 min) by apoptosis, as determined by the accumulation of pre-G1 apoptotic cells and the internucleosomal fragmentation of DNA. Addition of calyculin A or okadaic acid (specific serine/threonine phosphatase inhibitors) or zinc chloride (apoptosis inhibitor) prevented the G1 arrest- and apoptosis-specific RB dephosphorylation. The levels of cyclin E- and cyclin A-associated kinase activities remained high during RB dephosphorylation, supporting the involvement of a chemotherapy-induced serine/threonine phosphatase(s) rather than p21. Furthermore, the induced phosphatase activity coimmunoprecipitated with the hyperphosphorylated RB and was active in a cell-free system that reproduced the growth arrest- and apoptosis-specific RB dephosphorylation, which was inhibitable by calyculin A but not zinc. We propose that the RB phosphatase(s) might be one of the p53-independent G1 checkpoint regulators.

Increased activity of p53 in senescing fibroblasts.

The p53 tumor-suppressor protein binds DNA and activates the expression of a 21-kDa protein that inhibits both the activity of cyclin-dependent kinases and the function of proliferating cell nuclear antigen. Since p21 expression has been reported to increase 10- to 20-fold as human diploid fibroblasts lose the ability to replicate, we examined the expression and activity of p53 during replicative aging. Similar levels of total p53 mRNA and protein were expressed in low-passage (young) and high-passage (old) cells but both DNA binding activity in vitro and transcriptional activity of p53 in vivo were increased severalfold in high-passage cells. While the basis of increased p53 activity is presently unclear, it is not correlated with differential phosphorylation or changes in p53-mouse double minute 2 gene product interactions. These results provide evidence for the activation of a protein involved in the control of cell cycle checkpoints during cellular aging, in the absence of increased expression.

p53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts.

Increased expression of wild-type p53 in response to DNA damage arrests cells late in the G1 stage of the cell cycle by stimulating the synthesis of inhibitors of cyclin-dependent kinases, such as p21/WAF1. To study the effects of p53 without the complication of DNA damage, we used tetracycline to regulate its expression in MDAH041 human fibroblasts that lack endogenous p53. When p53 is expressed at a level comparable to that induced by DNA damage in other cells, most MDAH041 cells arrested in G1, but a significant fraction also arrested in G2/M. Cells released from a mimosine block early in S phase stopped predominantly in G2/M in the presence of p53, confirming that p53 can mediate arrest at this stage, as well as in G1. In these cells, there was appreciable induction of p21/WAF1. MDAH041 cells arrested by tetracycline-regulated p53 for as long as 20 days resumed growth when the p53 level was lowered, in striking contrast to the irreversible arrest mediated by DNA damage. Therefore, irreversible arrest must involve processes other than or in addition to the interaction of p53-induced p21/WAF1 with G1 and G2 cyclin-dependent kinases.

The yeast nuclear import receptor is required for mitosis.

The nuclear import system is highly conserved among eukaryotes. Here we report the effects of a conditional mutation in SRP1, which encodes a Saccharomyces cerevisiae homolog of the vertebrate nuclear import receptor importin. Importin was isolated as a factor required for the initial targeting step of a nuclear import substrate to the nuclear envelope in a mammalian in vitro assay. We show that yeast Srp1 is similarly required for protein import. In addition, Srp1 is also required for the execution of mitosis: we demonstrate that cells containing a conditional mutation of SRP1 arrest with a G2/M phenotype in a manner analogous to classic cdc mutants. This defect may be due to the failure of the mutant to degrade the mitotic cyclin Clb2 and other proteins required for mitosis. The requirement of a nuclear import receptor for cell cycle-regulated proteolysis implies that import of cell cycle regulators into the nucleus is critical for cell cycle progression.

Identification of a plant serine-arginine-rich protein similar to the mammalian splicing factor SF2/ASF.

We show that the higher plant Arabidopsis thaliana has a serine-arginine-rich (SR) protein family whose members contain a phosphoepitope shared by the animal SR family of splicing factors. In addition, we report the cloning and characterization of a cDNA encoding a higher-plant SR protein from Arabidopsis, SR1, which has striking sequence and structural homology to the human splicing factor SF2/ASF. Similar to SF2/ASF, the plant SR1 protein promotes splice site switching in mammalian nuclear extracts. A novel feature of the Arabidopsis SR protein is a C-terminal domain containing a high concentration of proline, serine, and lysine residues (PSK domain), a composition reminiscent of histones. This domain includes a putative phosphorylation site for the mitotic kinase cyclin/p34cdc2.

14-3-3 proteins associate with cdc25 phosphatases.

The cdc25 phosphatases play key roles in cell cycle progression by activating cyclin-dependent kinases. Two members of the 14-3-3 protein family have been isolated in a yeast two-hybrid screen designed to identify proteins that interact with the human cdc25A and cdc25B phosphatases. Genes encoding the human homolog of the 14-3-3 epsilon protein and the previously described 14-3-3 beta protein have been isolated in this screening. 14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that were originally isolated in mammalian brain preparations and that possess diverse biochemical activities related to signal transduction. We present evidence that indicates that cdc25 and 14-3-3 proteins physically interact both in vitro and in vivo. 14-3-3 protein does not, however, affect the phosphatase activity of cdc25A. Raf-1, which is known to bind 14-3-3 proteins, has recently been shown to associate with cdc25A and to stimulate its phosphatase activity. 14-3-3 protein, however, has no effect on the cdc25A-kinase activity of Raf-1. Instead, 14-3-3 may facilitate the association of cdc25 with Raf-1 in vivo, participating in the linkage between mitogenic signaling and the cell cycle machinery.

Diffusion and formation of microtubule asters: physical processes versus biochemical regulation.

Microtubule asters forming the mitotic spindle are assembled around two centrosomes through the process of dynamic instability in which microtubules alternate between growing and shrinking states. By modifying the dynamics of this assembly process, cell cycle enzymes, such as cdc2 cyclin kinases, regulate length distributions in the asters. It is believed that the same enzymes control the number of assembled microtubules by changing the "nucleating activity" of the centrosomes. Here we show that assembly of microtubule asters may be strongly altered by effects connected with diffusion of tubulin monomers. Theoretical analysis of a simple model describing assembly of microtubule asters clearly shows the existence of a region surrounding the centrosome depleted in GTP tubulin. The number of assembled microtubules may in some cases be limited by this depletion effect rather than by the number of available nucleation sites on the centrosome.

Rescue of defective mitogenic signaling by D-type cyclins.

Three gene products, including Myc and the D- and E-type G1 cyclins, are rate limiting for G1 progression in mammalian fibroblasts. Quiescent mouse NIH 3T3 fibroblasts engineered to express a mutant colony-stimulating factor (CSF-1) receptor (CSF-1R 809F) fail to synthesize c-myc and cyclin D1 mRNAs upon CSF-1 stimulation and remain arrested in early G1 phase. Ectopic expression of c-myc or either of three D-type cyclin genes, but not cyclin E, resensitized these cells to the mitogenic effects of CSF-1, enabling them to proliferate continuously in liquid culture and to form colonies in agar in response to the growth factor. Rescue by cyclin D1 was enhanced by c-myc but not by cyclin E and was reversed by infecting cyclin D1-reconstituted cells with a retroviral vector encoding catalytically inactive cyclin-dependent kinase 4. Induction of cyclin D1 mRNA by CSF-1 was restored in cells forced to express c-myc, and vice versa, suggesting that expression of the two genes is interdependent. Cells reconstituted with c-myc were prevented from entering S phase when microinjected with a monoclonal antibody to cyclin D1, and conversely, those rescued by cyclin D1 were inhibited from forming CSF-1-dependent colonies when challenged with a dominant-negative c-myc mutant. Cyclin D mutants defective in binding to the retinoblastoma protein were impaired in rescuing mitogenic signaling. Therefore, Myc and D-type cyclins collaborate during the mitogenic response to CSF-1, whereas cyclin E functions in a separate pathway.

Growth suppression by p16ink4 requires functional retinoblastoma protein.

p16ink4 has been implicated as a tumor suppressor that is lost from a variety of human tumors and human cell lines. p16ink4 specifically binds and inhibits the cyclin-dependent kinases 4 and 6. In vitro, these kinases can phosphorylate the product of the retinoblastoma tumor suppressor gene. Thus, p16ink4 could exert its function as tumor suppressor through inhibition of phosphorylation and functional inactivation of the retinoblastoma protein. Here we show that overexpression of p16ink4 in certain cell types will lead to an arrest in the G1 phase of the cell cycle. In addition, we show that p16ink4 can only suppress the growth of human cells that contain functional pRB. Moreover, we have compared the effect of p16ink4 expression on embryo fibroblasts from wild-type and RB homozygous mutant mice. Wild-type embryo fibroblasts are inhibited by p16ink4, whereas the RB nullizygous fibroblasts are not. These data not only show that the presence of pRB is crucial for growth suppression by p16ink4 but also indicate that the pRB is the critical target acted upon by cyclin D-dependent kinases in the G1 phase of the cell cycle.

Cyclins as markers of tumor proliferation: immunocytochemical studies in breast cancer.

We have developed methods to use anticyclin A, B, and E antibodies as reagents to specifically detect proliferating cells in specific phases of the cell cycle in formalin-fixed, paraffin-embedded sections of tissues and cells. Staining of 48 archival cases of breast cancer showed that these antibodies estimate the tumor proliferation fraction and therefore are potentially useful for the prediction of prognosis. A subset of cancers had a high frequency of tumor cells expressing cyclins A and E, out of proportion to other proliferation markers, suggesting that these tumors may have deregulated cyclin expression. In addition to recognizing authentic cyclin E in the nucleus of proliferating cells, anticyclin E antibody cross-reacted with a cytoplasmic protein in nonproliferating endothelial cells. This cross-reaction allows the simultaneous visualization and quantitation of microvessels in the tumors, measuring a second potential predictor of breast cancer prognosis, tumor angiogenesis.

Involvement of the cell-cycle inhibitor Cip1/WAF1 and the E1A-associated p300 protein in terminal differentiation.

The mechanism of cell cycle withdrawal during terminal differentiation is poorly understood. We report here that the cyclin-dependent kinase (CDK) inhibitor p21Cip1/WAF1 is induced at early times of both keratinocyte and myoblast differentiation. p21Cip1/WAF1 induction is accompanied by a drastic inhibition of total Cdk2, as well as p21Cip1/WAF1-associated CDK kinase activities. p21Cip1/WAF1 has been implicated in p53-mediated G1 arrest and apoptosis. In keratinocyte differentiation, Cip1/WAF1 induction is observed even in cells derived from p53-null mice. Similarly, keratinocyte differentiation is associated with induction of Cip1/WAF1 promoter activity in both wild-type and p53-negative keratinocytes. Induction of the Cip1/WAF1 promoter upon differentiation is abolished by expression of an adenovirus E1A oncoprotein (d1922/947), which is unable to bind p105-Rb, p107, or cyclin A but which still binds the nuclear phosphoprotein p300. Overexpression of p300 can suppress the E1A effect, independent of its direct binding to E1A. Thus, terminal differentiation-induced growth arrest in both keratinocyte and myoblast systems is associated with induction of Cip1/WAF1 expression. During keratinocyte differentiation, Cip1/WAF1 induction does not require p53 but depends on the transcriptional modulator p300.

Biological response to phorbol ester determined by alternative G1 pathways.

A plethora of extracellular signals is known to induce a common set of immediate early genes. The immediate early response, therefore, must not be sufficient to determine the biological outcome. An example of this is found with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). A potent activator of protein kinase C, TPA can either stimulate or inhibit cell proliferation, depending on the cell type. This cell context-dependent response to TPA is observed with two subclones of NIH 3T3 cells, the P- and the N-3T3 clones. TPA is a mitogen for the P-3T3 but an antimitogen for the N-3T3 cells. The immediate early pathway is activated by TPA in both cell types, indicating that this pathway alone does not activate DNA synthesis. The delayed induction of cyclin D1 expression by TPA is observed only in the P-3T3 cells, correlating with mitogenesis. N-Acetylcysteine does not affect the immediate early pathway but can inhibit the TPA-mediated induction of cyclin D1 and DNA synthesis. In the N-3T3 cells, TPA causes an inhibition of the cyclin E-associated kinase at the G1/S transition, correlating with growth inhibition. The growth-inhibitory activity of TPA is not affected by N-acetylcysteine. Thus, the two TPA-regulated G1 pathways can be distinguished by their sensitivity to N-acetylcysteine. These results demonstrate that TPA can activate alternative G1 pathways. Moreover, the selection of the alternative G1 pathways is determined by the cell context, which, in turn, dictates the biological response to TPA.

A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription.

Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II has been suggested to be critical for transcription initiation, activation, or elongation. A kinase activity specific for CTD is a component of the general transcription factor TFIIH. Recently, a cyclin-dependent kinase-activator kinase (MO15 and cyclin H) was found to be associated with TFIIH preparations and was suggested to be the CTD kinase. TFIIH preparations containing mutant, kinase-deficient MO15 lack CTD kinase activity, indicating that MO15 is critical for polymerase phosphorylation. Nonetheless, these mutant TFIIH preparations were fully functional (in vitro) in both basal and activated transcription. These results indicate that CTD phosphorylation is not required for transcription with a highly purified system.