Combinatorial roles for pRB, p107, and p130 in E2F-mediated cell cycle control

Numerous studies have implicated the pRB family of nuclear proteins in the control of cell cycle progression. Although over-expression experiments have revealed that each of these proteins, pRB, p107, and p130, can induce a G(1) cell cycle arrest, mouse knockouts demonstrated distinct developmental requirements for these proteins, as well as partial functional redundancy between family members. To study the mechanism by which the closely related pRB family proteins contribute to cell cycle progression, we generated 3T3 fibroblasts derived from embryos that lack one or more of these proteins (pRB(-/-), p107(-/-), p130(-/-), pRB(-/-)/p107(-/-), pRB(-/-)/p130(-/-), and p107(-/-)/p130(-/-)). By comparing the growth and cell cycle characteristics of these cells, we have observed clear differences in the manner in which they transit through the G(1) and S phases as well as exit from the cell cycle. Deletion of Rb, or more than one of the family members, results in a shortening of G(1) and a lengthening of S phase, as well as a reduction in growth factor requirements. In addition, the individual cell lines showed differential regulation of a subset of E2F-dependent gene promoters, as well as differences in cell cycle-dependent kinase activity. Taken together, these observations suggest that the closely related pRB family proteins affect cell cycle progression through distinct biochemical mechanisms and that their coordinated action may contribute to their diverse functions in various physiological settings.     

p53-dependent G1 arrest involves pRB-related proteins and is disrupted by the human papillomavirus 16 E7 oncoprotein.

The cell cycle regulatory tumor suppressor proteins p53 and pRB are targeted for inactivation by several tumor viruses, including the high-risk types of human papillomaviruses (HPVs) via interactions of the HPV E6 and E7 oncoproteins with p53 and pRB, respectively. p53 plays a central role in a signal transduction pathway that mediates G1 arrest after DNA damage, though the mechanism by which G1 arrest occurs has not been elucidated. The cyclin-associated protein p21waf1/cip1 has recently been shown to be induced by p53 and to inhibit cyclin complex-mediated phosphorylation of pRB in vitro. Thus, we investigated a possible role for pRB in the p53-mediated DNA damage response. After gamma-irradiation, cells expressing wild-type p53 arrested in G1, contained increased levels of WAF1/CIP1 mRNA, and demonstrated accumulation of hypophosphorylated pRB. In contrast, cell lines with abnormal p53 genes or with p53 functionally inactivated by the E6 oncoprotein of HPV16 (a high-risk HPV) failed to arrest in G1, did not elevate WAF1/CIP1 mRNA, and did not accumulate hypophosphorylated pRB. Despite apparently normal elevation of p53 protein and WAF1/CIP1 mRNA after irradiation, cells expressing HPV16 E7 also failed to arrest in G1 and did not accumulate hypophosphorylated pRB. Disruption of RB genes alone did not totally abrogate this G1 arrest. Our results suggest that p53 indirectly regulates phosphorylation of pRB and that pRB and/or other pRB-like molecules that bind to HPV16 E7 participate in the DNA damage-mediated G1 arrest signal. In the process of HPV infection, the HPV E6 and E7 oncoproteins may undermine this cell cycle checkpoint, contributing to the accumulation of genetic alterations during tumorigenesis.     

Rapamycin inhibits human in stent restenosis vascular smooth muscle cells independently of pRB phosphorylation and p53

OBJECTIVE: Drug-eluting stents containing the immunosuppressant rapamycin markedly inhibit in stent restenosis (ISR). However, the molecular mechanisms that underlie its effect on ISR-derived vascular smooth muscle cells (VSMCs), as opposed to normal VSMCs, are unknown. Specifically, as ISR-VSMCs have altered cell cycle regulation, rapamycin may arrest these cells via novel molecular pathways. METHODS: We isolated human VSMCs from sites of ISR, and examined the effect of rapamycin on cell proliferation using MTT assay, time lapse videomicroscopy and flow cytometry. Regulation of G(1)-S transition was examined using Western blotting, and cell size and protein synthesis examined using flow cytometry and collagen assay, respectively. The requirement for pRB and p53 was examined using ISR VSMCs expressing E1A and a dominant negative p53, respectively. RESULTS: ISR-VSMC proliferation was potently inhibited by rapamycin. Arrest was confined to G(1), as cell proliferation (but not cell size) of S/G(2)-arrested cells was unaffected by rapamycin. Moreover, ISR-VSMC lines generated with disrupted p53 or pRB function still arrested in the presence of rapamycin, suggesting that these genes are dispensable for rapamycin-induced arrest. Significantly, rapamycin completely inhibited the phosphorylation of p70(S6K), an mTOR-regulated kinase implicated in the control of proliferation, but had no effect on collagen or total protein synthesis. CONCLUSIONS: We demonstrate that rapamycin is a potent inhibitor of ISR VSMC proliferation during G(1). Rapamycin's action does not require p53 or pRB. We show that p70(S6K) is markedly inhibited in rapamycin-arrested ISR cells, suggesting that regulation of its upstream kinase, mTOR, is important for the control of proliferation in ISR cells.     

Expression of thymidylate synthase in human gastric and colorectal adenocarcinomas is upregulated by p16/INK4

BACKGROUND/AIMS: We observed the relationship between the expression of thymidylate synthase protein (pTS) and cell cycle regulators in gastric and colorectal adenocarcinoma tissues. METHODOLOGY: This study included 80 gastric and 50 colorectal adenocarcinomas. Immunohistochemical staining was performed using a polyclonal antibody to recombinant human pTS, and monoclonal antibodies to p53, p21/WAF1CIP1, p16/INK4, cyclin D1 and pRB. Each staining was quantified using computerized image analysis on a CAS 200 system. We selected the mean expression values as the cutoff values to distinguish between high and low expression of these substances. RESULTS: There was no relationship between pTS expression and p21/WAF1CIP1, cyclin D1, or pRB expression in gastric and colorectal carcinomas. In both gastric and colorectal carcinomas, the pTS expression was significantly low in the high p16/INK4 expression subgroup compared with the low p16/INK4 expression subgroup (P < 0.05). Further, the pTS expression was significantly high in the high p53 expression subgroup compared with the low p53 expression subgroup in colorectal adenocarcinomas (P < 0.05). CONCLUSIONS: pTS expression regulation in human gastric and colorectal adenocarcinomas in complex, and upregulated by p16/INK4.     

Cyclin D1 overexpression in thyroid carcinomas: relation with clinico-pathological parameters, retinoblastoma gene product, and Ki67 labeling index.

Cyclin D1 is a G1 cyclin participating in the control of cell cycle progression through interaction with the retinoblastoma gene product (pRB). The overexpression of positive regulators (such as cyclin D1) has been reported in a variety of neoplasms, but their role in thyroid tumorigenesis is yet to be established. In our series of 54 thyroid carcinomas, cyclin D1 overexpression (detected by both immunohistochemistry and by Northern blotting) was correlated with prognostic variables, proliferative activity and pRB. Cyclin D1 overexpression was observed in 35% of thyroid carcinomas with a significantly higher expression of this cyclin in neoplastic tissues than in matched normal parenchyma. In well-differentiated carcinomas, the cyclin D1 mRNA overexpression was inversely correlated with nodal status (p = 0.03), while the protein product was higher in tumors from patients less than 40 than patients over 40 years of age. Inversely, there was no significant correlation with gender and tumor status, pRB and with proliferative activity.     

The molecular biology of pituitary tumors: a personal perspective

Oncogenes and tumor suppressor genes involved in most common cancers are not involved in the great majority of pituitary adenomas. Similarly, there is little evidence to suggest that the mutations involved in genetic syndromes associated with pituitary tumors (such as the gsp, MEN1, PKAR1A or AIP mutations) are common in sporadic tumors. A novel pituitary tumor transforming gene (PTTG, securin) has been identified which is over-expressed in most tumors—but it is unclear as to its causal role in oncogenesis. Cell signaling abnormalities have been identified in pituitary tumors but their genetic basis is unknown. However, both the Akt pathway and the MAPK pathway are over-expressed in many pituitary tumors, which results in the inhibition of cell cycle inhibitors. These pathways share a common root in the tyrosine kinase receptor, and a change to these receptors or their relationship to membrane matrix-related proteins may be an early event in tumorigenesis.     

Estrogen action in the regulation of cell proliferation, cell survival, and tumorigenesis in the rat anterior pituitary gland

Estrogens act as important regulators of cell proliferation, cell survival, and differentiation in a variety of organ systems and tissues and have been implicated in the etiology of a variety of malignant cancers and benign tumors. The anterior pituitary gland of the rat provides an excellent model for the study of estrogen action in the regulation of cell proliferation and survival. Estrogens stimulate proliferation of the prolactin (PRL)-producing lactotroph and enhance lactotroph survival. Through these actions on lactotroph proliferation and survival, estrogens induce or contribute to the development of PRL-producing pituitary tumors in several rat strains. Data from our laboratory and others indicate that estrogen-induced pituitary growth is rat strain specific and segregates as a quantitative genetic trait in crosses between different rat strains. The purpose of this review is to summarize current know ledge pertaining to estrogen action in the regulation of cell proliferation, cell survival, and tumorigenesis in the anterior pituitary gland of the rat species, Rattus norvegicus, and to illustrate the advantages of the rat pituitary gland as a model for elucidating the mechanisms through which estrogens regulate these processes.     

Points of integration between the intracellular energy sensor AMP-activated protein kinase (AMPK) activity and the somatotroph axis function

AMP-activated protein kinase (AMPK), an enzyme functioning as a cellular sensor of low energy, stores and promotes adaptive changes in growth, differentiation, and metabolism. While AMPK is primarily thought of as a regulator of systemic metabolism, it has been clearly established that it also has a role in the regulation of cell growth and may be a therapeutic target for proliferative disorders. Growth hormone (GH) secretion from the anterior pituitary and GH-induced synthesis and release of insulin-like-growth-factor-1 (IGF-1) from the liver determine linear growth before puberty. Actually, GH and IGF-1 are potent growth factors affecting cell growth and differentiation in different tissues, and still have anabolic functions and serve as essential regulators of fuel metabolism in adulthood, as well. A variety of peripheral hormonal and metabolic signals regulate GH secretion either by acting directly on the anterior pituitary and/or modulating GH-releasing hormone or somatostatin release from the hypothalamus. Actually, intracellular transduction of endocrine and metabolic signals regulating somatotroph function is still debated. Based on the previously summarized contents, the aim of the present work has been to review currently available data suggesting a role of AMPK in the interplay between GH axis activity and metabolic functions.     

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.     

Genetics of human stature: Insight from single gene disorders

Mutations of numerous genes encoding proteins that affect multiple pathways responsible for regulation of cell proliferation can cause growth disturbances in humans. Genes such as HESX1, PROP1, PIT1/POU1F1 and GLI2 have been shown to cause pituitary hormone deficiency. In addition, heterozygous mutations or gene deletions in the growth hormone-insulin-like growth factor (GH-IGF) axis such as the GH, GH-releasing hormone receptor, GH receptor, STAT5b, IGF-I, IGF-I receptor and the acid labile subunit have also been observed in children with growth failure and short stature. More recently, mutations of genes encoding regulators of cell proliferation and division, i.e., the pericentrin gene, have also resulted in severe growth disturbances.     

Development and regulation of growth and differentiated function in human and subhuman primate fetal gonads

We have attempted to summarize the research on primate fetal gonadal development that has occurred over the past three decades. Many similarities exist between fetal gonadal development in human and subhuman primates; therefore, comparisons and analogies between these species can be made. Fetal gonadal development is a complex process dependent on timely maturation and differentiation of several cell types with different functions. Adequate development is important for normal sexual development and intact adult fertility potential as well as for intrauterine priming of neural centers in the central nervous system. While the fetal primate testis is active in steroidogenesis, the fetal ovary seems to be quiescent throughout most of gestation, although some ovarian steroidogenic enzymes have been demonstrated. Growth and development of both gonads are controlled during late gestation at least in part by pituitary hormones, while earlier in gestation other yet undefined regulators (placental, intragonadal) likely also are active. The main goal of this review was to demonstrate that gonadal growth and differentiation, both in males and females, is regulated by endocrine factors as well as by intragonadal, autocrine/paracrine agents. Although many parts of the puzzle are still missing it is probable that, similar to fetal development of other endocrine tissues and to events in postnatal gonads, these local regulators have important functions. Currently, primate fetal gonadal research is lacking in at least two key aspects: 1) the definition of paracrine and autocrine nonsteroidal factors that are involved in the regulation of gonadal growth and differentiation in vitro; and 2) in vivo studies in subhuman primates that might better help to clarify the biological roles of the multiple extra- and intragonadal hormones and their complex interactions. To date, the regulation of gonadal steroidogenesis has been investigated more thoroughly than the regulation of gonadal growth. Most of our knowledge stems from observations of gonadal development in anencephalics or subhuman primates after pituitary ablation. Because of the constraints of small organ size and limitation of material, studies of fetal primate gonadal development have been limited. Given such limitations, new molecular biological techniques, including polymerase chain reaction and in situ hybridization, may provide the means of addressing these questions. Further, because of these limitations, sensitive cell separation techniques need to be developed to achieve enriched primary gonadal cell cultures from individual gonads.