Rb Target Research Articles

Rb Enhances p160/SRC Coactivator-dependent Activity of Nuclear Receptors and Hormone Responsiveness

The retinoblastoma tumor suppressor protein (Rb) is best known as a repressor of genes involved in cell cycle progression. Rb has also been implicated in activation of transcription, in particular by nuclear receptors (NRs) and by differentiation-related transcription factors, but the relevance of this activity is unclear. We show that Rb and the related proteins p107 and p130 enhance the activity of NRs related to NGFI-B (Nur factors) through direct interactions with NGFI-B and SRC-2. Although recruitment of SRC/p160 coactivators to the NGFI-B AF1 domain is independent of Rb, its presence enhances SRC-dependent transcription. Rb potentiation of SRC coactivators is exerted on a subset (Nur factors, hepatocyte nuclear factor-4 (HNF-4), SF-1, and ER) but not all NRs. The levels of Rb-related proteins modulate hormone responsiveness of the NGFI-B-dependent pituitary proopiomelanocortin gene and HNF-4-dependent transcription during enterocyte differentiation. Increased Rb expression upon cell differentiation may promote differentiated functions, at least in part, by potentiation of NR activity.

S‐phase entry of oligodendrocyte lineage cells is associated with increased levels of p21Cip1

The mechanisms regulating the number of myelinating cells in the central nervous system are crucial for both normal development and repair in pathological conditions. Among relevant growth factors involved in this process, fibroblast growth factor-2 (FGF2) induces oligodendrocyte progenitors (OLPs) to proliferate and stimulates mature oligodendrocytes (OLs) to reenter the S-phase of the cell cycle. S-phase entry is modulated by the formation of complexes between cyclins and cyclin-dependent kinases (CDKs), on one hand, and by their interactions with cell cycle inhibitors (e.g., p18INK, p27Kip1, p21Cip1), on the other. Although the roles of cyclin E/CDK2 complexes and the inhibitor p27Kip1 have been extensively investigated relative to proliferation and differentiation in the OL lineage, less is known about the regulation of the formation of cyclin D1/CDK4 complexes and the role of p21Cip1 in these events. In this study, we show that the FGF2-mediated increase in bromodeoxyuridine (BrdU) incorporation into OL progenitors and mature OLs occurs concomitantly with increase in the levels of p21Cip1 and the formation of p21Cip1/cyclin D1/CDK4 ternary complexes. These complexes are functionally active is indicated by the ensuing FGF2-dependent hyperphosphorylation of the downstream target Rb. In untreated mature OLs that do not incorporate BrdU, the levels of p21Cip1 are low, and the level of the inhibitor p18INK is high. Furthermore, p18INK sequesters CDK2 into binary complexes, precluding the formation of p21Cip1/cyclin D1/CDK4 ternary complexes in these cells. Therefore, we propose that p21Cip1 is acting as a positive regulator, rather than an inhibitor, of cell cycle entry by favoring the assembly of active cyclin D1/CDK4 complexes.

Differential role of RB in response to UV and IR damage

The retinoblastoma tumor suppressor (RB) is functionally inactivated in the majority of cancers and is a critical mediator of DNA damage checkpoints. Despite the critical importance of RB function in tumor suppression, the coordinate impact of RB loss on the response to environmental and therapeutic sources of damage has remained largely unexplored. Here, we utilized a conditional knockout system to ablate RB in adult fibroblasts. This model system enabled us to investigate the temporal role of RB loss on cell cycle checkpoints and DNA damage repair following ultraviolet (UV) and ionizing radiation (IR) damage. We demonstrate that RB loss compromises rapid cell cycle arrest following UV and IR exposure in adult primary cells. Detailed kinetic analysis of the checkpoint response revealed that disruption of the checkpoint is concomitant with RB target gene deregulation, and is not simply a manifestation of chronic RB loss. RB loss had a differential effect upon repair of the major DNA lesions induced by IR and UV. Whereas RB did not affect resolution of DNA double-strand breaks, RB-deficient cells exhibited accelerated repair of pyrimidine pyrimidone photoproducts (6-4 PP). In parallel, this repair was coupled with enhanced expression of specific factors and the behavior of proliferating cell nuclear antigen (PCNA) recruitment to replication and repair foci. Thus, RB loss and target gene deregulation hastens the repair of specific lesions distinct from its ubiquitous role in checkpoint abrogation.

The HTLV-I Tax oncoprotein targets the retinoblastoma protein for proteasomal degradation

Human T-cell leukemia virus type-I (HTLV-I), the etiologic agent of adult T-cell leukemia (ATL), is estimated to affect 10-20 million people worldwide. The transforming ability of HTLV-I has been largely attributed to the viral protein Tax, which modulates the activity of several well-known cell cycle regulators. An important cell cycle regulator, the retinoblastoma (Rb) protein, is often inactivated in many cancers including virally induced cancers. Upon examination of Rb status, we observed a decrease in Rb protein expression in HTLV-1-infected cell lines as well as in ex vivo ATL patient samples. Transient transfection assays indicated that decreased Rb protein levels were Tax dependent. Here, we demonstrate for the first time that Tax directly associates with Rb. This interaction was localized within the B pocket of Rb and the C-terminus of Tax (aa 245-353). Within the C-terminus of Tax, we have identified an LXCXE-like motif, that when mutated resulted in the loss of Tax/Rb interaction. Furthermore, through the use of proteasome inhibitors, such as MG-132, in vivo and proteasome degradation assays in vitro, we found that Tax destabilizes the hypo-phosphorylated (active) form of Rb via the proteasome pathway. Therefore, we propose a model whereby Tax targets Rb to the proteasome by acting as a molecular bridge bringing Rb into contact with the proteasome for degradation.

Apoptotic and mitogenic stimuli inactivate Rb by differential utilization of p38 and cyclin-dependent kinases.

Inactivation of the retinoblastoma (Rb) tumor suppressor protein is essential for the G1/S transition during mammalian cell cycle progression. Although Rb is inactivated by phosphorylation by cyclins D and E and their associated kinases during cell cycle progression, we find that Rb is inactivated upon apoptotic stimulation by Fas through the mediation of p38 kinase, independent of cyclins and cyclin-dependent kinases (cdks). Inactivation by p38 kinase coincided with increased phosphorylation of Rb leading to dissociation of E2F and increased transcriptional activity; such p38-mediated changes in Rb function occurred only during Fas stimulation but not mitogenic progression. p38 kinase targets Rb preferentially and had minimal effects on p107 and had no effect on p130 function. We also find that phosphorylation site mutants of Rb (PSM7LP and PSM9-Rb) that cannot be inactivated by cdks can be targeted by Fas and p38 kinase, suggesting that Rb inactivation by these kinases is biochemically and functionally distinct. It appears that Rb inactivation is achieved by different kinase cascades in response to mitogenic and apoptotic signals.

Telomerase activation by human papillomavirus type 16 E6 protein: induction of human telomerase reverse transcriptase expression through Myc and GC-rich Sp1 binding sites.

High-risk human papillomaviruses (HPVs) immortalize keratinocytes by disrupting the retinoblastoma protein (Rb)/p16 pathway and activating telomerase. The E7 oncoprotein targets Rb, while the E6 oncoprotein induces telomerase activity in human keratinocytes. This study has examined the mechanism by which E6 activates telomerase. Expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, was found to be increased in keratinocytes stably expressing HPV type 16 E6, suggesting that E6 acts to increase hTERT transcription. hTERT expression and telomerase activity were activated to significantly higher levels in cells expressing both E6 and E7 than in cells expressing E6 alone. This indicates that E7 may augment E6-mediated activation of hTERT transcription. In transient-transfection assays using hTERT reporters, the induction of hTERT expression by E6 was found to be mediated by a 258-bp fragment of the hTERT promoter, proximal to the ATG initiation codon. Previous studies have demonstrated that overexpression of Myc can activate hTERT expression, suggesting that Myc may be a mediator of E6-mediated hTERT induction. However, in cells stably expressing E6, no strict correlation between the level of Myc and the activation of hTERT was found. Consistent with this observation, mutation of the two Myc binding sites in the hTERT promoter only modestly reduced responsiveness to E6 in transient reporter assays. This indicates that activation of Myc-dependent transcription is not essential for E6-mediated upregulation of hTERT expression. The hTERT promoter also contains five GC-rich elements that can bind Sp1. Mutation of these sites within the 258-bp fragment partially reduced hTERT induction by E6. However, when mutations in the Sp1 sites were combined with the mutated Myc binding sites, all activation by E6 was lost. This indicates that it is the combinatorial binding of factors to Myc and Sp1 cis elements that is responsible for hTERT induction by E6.

Depth of origin of atoms sputtered from crystalline targets

Recently, V.I. Shulga and W. Eckstein (Nucl. Instr. and Meth. B 145 (1998) 492) investigated the depth of origin of atoms sputtered from random elemental targets using the Monte Carlo code TRIM.SP and the lattice code OKSANA. They found that the mean depth of origin is proportional to N −0.86, where N is the atomic density; and that the most probable escape depth is ∼ λ 0/2, where λ 0 is the mean atomic distance. Since earlier molecular dynamics simulations with small crystalline elemental targets typically produced a most probable escape depth of zero (i.e., most sputtered atoms came from the topmost layer of the target), we have carried out new molecular dynamics simulations of sputtered atom escape depths with much larger crystalline targets. Our new results, which include the bcc targets Cs, Rb and W, as well as the fcc targets Cu and Au predict that the majority of sputtered atoms come from the first atomic layer for the bcc(1 0 0), bcc(1 1 1), fcc(1 0 0) and fcc(1 1 1) targets studied. For the high-atomic density targets Cu, Au and W, the mean depth of origin of sputtered atoms typically is less than 0.25 λ 0. For the low-atomic density targets Cs and Rb, the mean depth of origin of sputtered atoms is considerably larger, and depends strongly on the crystal orientation. We show that the discrepancy between the single-crystal and amorphous target depth of origin values can be resolved by applying a simple correction to the single-crystal results.

R b activation and repression of c- myc transcription precede apoptosis of human prostate epithelial cells

Objectives. The retinoblastoma gene (Rb) encodes a transcriptional regulatory protein that functions in the regulation of cellular growth, differentiation, and survival. Many of the target genes of Rb with respect to growth regulation and differentiation have been identified. However, the identities of the Rb target genes involved in the regulation of cell survival and cell death (apoptosis) are unknown. We sought to determine whether the c -myc oncogene, a known target of Rb activity in cell cycle control, is also recruited in an apoptotic pathway uniquely regulated by Rb in prostate epithelial cells. Methods. We previously described a cell culture model to study the apoptosis of prostate cancer cells in which the human prostate cancer line, LNCaP, will undergo apoptosis after inducible expression and activation of the alpha isozyme of protein kinase C (PKC) or after exposure to low concentrations of the PKC activator TPA. Rb protein and c- myc mRNA and protein were evaluated in the Rb+/+ LNCaP and in the Rb−/− DU145 prostate cancer cells. Results. TPA-induced apoptosis in LNCaP cells was preceded by the rapid depletion of c- myc mRNA. The DU145 cultures were resistant to TPA-induced apoptosis and the c- myc mRNA levels remained elevated. The examination of Rb protein in the LNCaP cells revealed rapid dephosphorylation preceding both c- myc protein depletion and apoptosis. Additionally, pretreatment of LNCaP cells with staurosporine, a potent inhibitor of several isozymes of the PKC family, inhibited apoptosis in these cells and completely blocked activation of Rb and repression of c- myc. Conclusions. On the basis of these studies, we suggest that induction of PKC-mediated apoptosis of Rb+/+ prostate cancer cells occurs by means of an intracellular pathway that involves the activation of Rb and repression of c- myc transcription.

RB regulates transcription of the p21/WAF1/CIP1 gene.

We have previously shown that RB plays an important role in the maintenance of the epithelial phenotype. p21 is also involved in several terminal differentiation systems including keratinocytes. We report here that p21 is an RB target gene in epithelial cells, but not in fibroblasts where RB is unable to transactivate p21 transcriptional expression. In epithelial cells, when RB family factors were inactivated by SV40 T antigen (LT), p21 expression was strongly repressed, whereas its expression was not affected when the cells were transformed by a mutated LT leaving RB active but inactivating p53. Moreover, retransformation by RB of LT transformed epithelial cells totally restored p21 expression. By cotransfection experiments and using deletions and point mutations of the p21 promoter, we show that the minimal region required for the RB-mediated transcriptional activation maps to a GC-rich region located between -83 and -74. This region is shown to interact specifically with the transcription factor Sp1 and Sp3. Thus for the first time, we show a positive transcriptional relationship between RB and p21 in epithelial cells. Since p21 keeps RB in a hypophosphorylated state important for its transcriptional activity during differentiation, our results imply an auto-loop of regulation between RB and p21 that may be essential for the maintenance of the differentiation state. We propose that this transcriptional relationship might be necessary of their roles in cell cycle arrest and in several differentiation pathways.

The plant cell cycle : G1/S regulation

G1/S controls appear central to the commitment to further cell division or differentiation in eukaryotic cells. Accumulating evidence implicates plant D-type cyclins as integrators of environmental and developmental signals within the cell cycle progression. The recent identification of plant homologs of retinoblastoma proteins (Rb) and E2F transcription factors strongly suggests that plants may deploy the E2F/Rb pathway in G1/S transition control in a similar manner as in mammalian cells. Overall, a picture of regulation is emerging: upon mitogenic signal stimulation,D-type cyclins are produced and associate with cyclin-dependent kinases (CDKs); the resulted CycD/CDK complexes phosphorylate subsequently the downstream Rb target proteins,leading to the release of free and transcriptionally active E2F factors from E2F/Rb complexes; the released E2F factors further promote transcription of genes whose products are required for G1/S transition as well as for Sphase progression. Potential roles of ubiquitin-dependent proteolysis in G1/S controls are also discussed.

Id2 Required for Myc Signals

The helix-loop-helix protein family member Id2 has been implicated in regulation of cell proliferation, differentiation, and apoptosis. Id2 is thought to act by interacting with basic helix-loop-helix transcription factors and inhibiting their interaction with DNA. Lasorella et al . provide new insight into how cell proliferation is influenced by Id2. They show that Id2 is required for oncoproteins of the Myc family to cause cell proliferation. Neuroblastoma cells often contain extra copies of the N- myc gene, and Lasorella et al . show that the Id2 gene is a direct target of N-Myc. The excess Id2 produced in neuroblastoma cells appears to sequester the active form of tumor supressor retinoblastoma protein (Rb). Disruption of the Rb pathway is characteristic of cancer cells, and overexpression of Myc or Id2 provides a potential mechanism by which this can occur without alterations in the Rb pathway itself. When normal amounts of Id2 are present in cells, Id2 appears to act as a target of Rb. Animals that lack Rb die during embryogenesis, but Lasorella et al . report that Id2-deficient mice develop further before dying at birth. Rb, in this case, may inhibit the action of Id2 on its normal targets. Thus loss of Id2 can rescue phenotypic effects of the loss of Rb. Lasorella, A., Noseda, M. Beyna, M., and Iavarone, A. (2000) Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins. Nature 407 : 592-598. [Online Journal]

RbAp48 Belongs to the Histone Deacetylase Complex That Associates with the Retinoblastoma Protein

The retinoblastoma susceptibility gene product, the Rb protein, is a key regulator of mammalian cell proliferation. One of the major targets of Rb is the S phase inducing E2F transcription factor. Once bound to E2F, Rb represses the expression of E2F-regulated genes. Transcriptional repression by Rb is believed to be crucial for the proper control of cell growth. Recently, we and others showed that Rb represses transcription through the recruitment of a histone deacetylase. Interestingly, we show here that the Rb-associated histone deacetylase complex could deacetylate polynucleosomal substrates, indicating that other proteins could be present within this complex. The Rb-associated protein RbAp48 belongs to many histone deacetylase complexes. We show here that the histone deacetylase HDAC1 is able to mediate the formation of a ternary complex containing Rb and RbAp48. Moreover, less deacetylase activity was found associated with Rb in cell extracts depleted for RbAp48 containing complexes, demonstrating that Rb, histone deacetylase, and RbAp48 are physically associated in live cells. Taken together, these data indicate that RbAp48 is a component of the histone deacetylase complex recruited by Rb. Finally, we found that E2F1 and RbAp48 are physically associated in the presence of Rb and HDAC1, suggesting that RbAp48 could be involved in transcriptional repression of E2F-responsive genes.

Cyclin A Is a Functional Target of Retinoblastoma Tumor Suppressor Protein-mediated Cell Cycle Arrest

Although RB inhibits the G(1)-S transition, the mechanism through which RB prevents cell cycle advancement remains unidentified. To delineate the mechanism(s) utilized by RB to exert its anti-proliferative activity, constitutively active RB proteins (which cannot be inactivated by phosphorylation) or p16ink4a (which prevents RB inactivation) were utilized. Both proteins inhibited the G(1)-S transition, whereas wild-type RB did not. We show that active RB acts to attenuate cyclin A promoter activity, and that overexpression of cyclin E reverses RB-mediated repression of the cyclin A promoter. Although cyclin A is an E2F-regulated gene, and it has been long hypothesized that RB mediates cell cycle advancement through binding to E2F and attenuating its transactivation potential, cyclin E does not reverse dominant negative E2F-mediated repression of the cyclin A promoter. Although active RB repressed both cyclin A and two other paradigm E2F-regulated promoters, only cyclin A transcription was restored upon co-expression of cyclin E. Additionally, we show that RB but not dominant negative E2F regulates the cyclin A promoter through the CCRE element. These data identify cyclin A as a downstream target of RB-mediated arrest. Consistent with this idea, ectopic expression of cyclin A reversed RB-mediated G(1) arrest. The findings presented suggest a pathway wherein cyclin A is a downstream target of RB, and cyclin E functions to antagonize this aspect of RB-mediated G(1)-S inhibition.

Neural precursor cells differentiating in the absence of Rb exhibit delayed terminal mitosis and deregulated E2F 1 and 3 activity.

The severe neurological deficit in embryos carrying null mutations for the retinoblastoma (Rb) gene suggests that Rb plays a crucial role in neurogenesis. While developing neurons undergo apoptosis in vivo neural precursor cells cultured from Rb-deficient embryos appear to differentiate and survive. To determine whether Rb is an essential regulator of the intrinsic pathway modulating terminal mitosis we examined the terminal differentiation of primary cortical progenitor cells and bFGF-dependent neural stem cells derived from Rb-deficient mice. Although Rb -/- neural precursor cells are able to differentiate in vitro we show that these cells exhibit a significant delay in terminal mitosis relative to wild-type cells. Furthermore, Rb -/- cells surviving in vitro exhibit an upregulation of p107 that is found in complexes with E2F3. This suggests that p107 may partially compensate for the loss of Rb in neural precursor cells. Functional ablation of Rb family proteins by adenovirus-mediated delivery of an E1A N-terminal mutant results in apoptosis in Rb-deficient cells, consistent with the interpretation that other Rb family proteins may facilitate differentiation and survival. While p107 is upregulated and interacts with the putative Rb target E2F3 in neural precursor cells, our results indicate that it clearly cannot restore normal E2F regulation. Rb-deficient cells exhibit a significant enhancement of E2F 1 and 3 activity throughout differentiation concomitant with the aberrant expression of E2F-inducible genes. In these studies we show that Rb is essential for the regulation of E2F 1 and 3 activity as well as the onset of terminal mitosis in neural precursor cells.

Identification of AP-2 as an interactive target of Rb and a regulator of the G1/S control element of the hamster histone H3.2 promoter.

Previous studies have established that a 32 bp cis -regulatory region, referred to as the H3core spanning -241 to -210 of the hamster histone H3.2 promoter, is critical for its G1/S-phase induction of transcription. Here we report that the transcription factor AP-2 is a major component of the protein complex which interacts with the H3core from hamster nuclear extracts. In search of the control mechanism(s) whereby AP-2 can mediate cell cycle regulation of the histone H3.2 promoter, we found that AP-2 can physically interact with the retinoblastoma (Rb) tumor suppressor protein in vitro , and when over-expressed, can also associate with Rb in vivo . Importantly, in contrast to the majority of Rb binding proteins, the C-terminal domain of Rb alone is sufficient for its interaction with AP-2. Using a reporter gene system linking tandem copies of the H3core to a heterologous minimal promoter, we demonstrated that over-expression of AP-2 proteins results in transactivation of the reporter gene through the H3core in a sequence-specific but orientation-independent manner. Additionally, this stimulative effect was suppressed by co-expression of Rb. Thus, AP-2, through its physical and functional interaction with Rb, may contribute to the cell cycle regulation of its target genes.

Human papillomavirus type 16 E7 protein sensitizes cervical keratinocytes to apoptosis and release of interleukin-1alpha.

Interleukin-1alpha (IL-1alpha) is a multifunctional cytokine that promotes inflammation, tissue remodeling and epithelial hyperplasia. Keratinocytes produce and sequester large amounts of biologically active IL-1alpha which can be released after injury or infection. We show that high level expression of human papillomavirus (HPV) type 16 E6 and E7 oncoproteins enhanced release of IL-1alpha from cultures of normal cervical keratinocytes (relative effectiveness E7 > E6/E7 >> E6 > control). The amount of IL-1alpha released was directly related to the ability of E7 or E6/E7 to stimulate apoptosis. E7 proteins that bound the retinoblastoma protein (Rb) strongly (HPV-16 and -18) induced more IL-1alpha release than those that bound poorly (HPV-6 and an HPV-16 E7 24gly mutant). Furthermore, overexpression of the E2F-1 transcription factor, a downstream target of Rb, induced extensive apoptosis and IL-1alpha release. Apoptosis and IL-1alpha release in response to growth factor removal occurred in part through a p53-independent pathway as coexpression of E6 and downregulation of p53 did not prevent either response. Immunohistochemical analyses showed that IL-1alpha was expressed by keratinocytes in normal cervical epithelia, low and high grade dysplasias, and cervical carcinomas. However, HPV-16 E6/E7 RNA expression and apoptosis increased in parallel in proliferating keratinocytes in severe dysplasias and carcinomas suggesting that IL-1alpha release is associated with progression to high grade disease. Thus, high level expression of the HPV-16 E7 protein sensitizes keratinocytes to apoptosis which results in release of IL-1alpha.

Regulation of differentiation by HBP1, a target of the retinoblastoma protein.

Differentiation is a coordinated process of irreversible cell cycle exit and tissue-specific gene expression. To probe the functions of the retinoblastoma protein (RB) family in cell differentiation, we isolated HBP1 as a specific target of RB and p130. Our previous work showed that HBP1 was a transcriptional repressor and a cell cycle inhibitor. The induction of HBP1, RB, and p130 upon differentiation in the muscle C2C12 cells suggested a coordinated role. Here we report that the expression of HBP1 unexpectedly blocked muscle cell differentiation without interfering with cell cycle exit. Moreover, the expression of MyoD and myogenin, but not Myf5, was inhibited in HBP1-expressing cells. HBP1 inhibited transcriptional activation by the MyoD family members. The inhibition of MyoD family function by HBP1 required binding to RB and/or p130. Since Myf5 might function upstream of MyoD, our data suggested that HBP1 probably blocked differentiation by disrupting Myf5 function, thus preventing expression of MyoD and myogenin. Consistent with this, the expression of each MyoD family member could reverse the inhibition of differentiation by HBP1. Further investigation implicated the relative ratio of RB to HBP1 as a determinant of whether cell cycle exit or full differentiation occurred. At a low RB/HBP1 ratio cell cycle exit occurred but there was no tissue-specific gene expression. At elevated RB/HBP1 ratios full differentiation occurred. Similar changes in the RB/HBP1 ratio have been observed in normal C2 differentiation. Thus, we postulate that the relative ratio of RB to HBP1 may be one signal for activation of the MyoD family. We propose a model in which a checkpoint of positive and negative regulation may coordinate cell cycle exit with MyoD family activation to give fidelity and progression in differentiation.

New perspectives on retinoblastoma family functions in differentiation.

Cell differentiation is a coordinated process that includes cell cycle exit and the expression of unique genes to specify tissue identity. The focus of this review is the recent progress in understanding the functions of the RB family (RB, p130,p107) in cell differentiation. Much work has focused on the functions of RB in G1 regulation. However, much evidence now suggests a diverse function in differentiation. For discussion, differentiation will be divided into three general steps: cell cycle exit, apoptosis protection, and tissue-specific gene expression. These processes are coordinated to provide the final and unique tissue characteristics. The RB family and targets such as E2F and HBP1 have functions in each step. While there is much knowledge on each separate step of differentiation, the mechanisms that coordinate cell cycle and tissue-specific events are still not known. New evidence suggests that this coordination contains both positive and negative regulation of tissue-specific gene expression. RB. p130, HBP1, and other proteins appear to have unexpected functions in regulating tissue-specific gene expression. The ubiquitous expressions of these proteins suggest membership in a new and general pathway to coordinate cell cycle events with tissue-specific gene expression during differentiation. The collective observations hypothesize the existence of a differentiation checkpoint to insure fidelity.

Optical pumping of thick alkali vapor for high-intensity polarized ion source

One-dimensional calculation of the polarization distribution in an alkali cell is applied to examine the optical pumping of a thick Rb target. Attenuation of pumping laser power is calculated in a long Rb cell by considering the radiation trapping. The results of the calculation for realistic laser power agree with experimental data of the mean value of the polarization in the cell. The effect of the attenuation on the polarization is clarified to relate to not only the thickness of the vapor. The results show that the maximum Rb thickness, in which a fixed polarization is achieved, is approximately proportional to the square root of the length of the cell.

Involvement of p21(WAF1/Cip1), CDK4 and Rb in activin A mediated signaling leading to hepatoma cell growth inhibition.

Cytokines are growth inhibitory in a target cell specific manner. The signaling pathways that characterize each cell type play a crucial role in determining the responsiveness to cytokine triggering. Activin A has been shown to suppress the growth of primary hepatocytes. Similarly, the human HepG2 hepatoma cell line was growth arrested by activin A as judged by lack of cell proliferation and suppression of DNA synthesis. In HepG2 cells activin A further induced accumulation of retinoblastoma protein in the hypophosphorylated form known to prevent entrance into S phase. This finding implies the involvement of cyclin dependent kinases and CDK inhibitors. Examination of HepG2 cells following addition of activin A revealed reduced expression of CDK4 and conversely, an increase in the CKI p21(WAF1/Cip1). This accumulation of p21(WAF1/Cip1) protein was partly due to increased transcriptional activity. Functional inactivation of p53, using a miniprotein that oligomerizes with p53 and abrogates DNA binding, abolished the ability of activin A to induce transcriptional activation from the p21(WAF1/Cip1) promoter. Thus, activin A, like transforming growth factor beta, seems to suppress cell growth through the downstream target Rb. However, each of these cytokines seem to operate through a distinct pathway.