Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d

The crystal structure of the cyclin D-dependent kinase Cdk6 bound to the p19 INK4d protein has been determined at 1.9 A resolution. The results provide the first structural information for a cyclin D-dependent protein kinase and show how the INK4 family of CDK inhibitors bind. The structure indicates that the conformational changes induced by p19INK4d inhibit both productive binding of ATP and the cyclin-induced rearrangement of the kinase from an inactive to an active conformation. The structure also shows how binding of an INK4 inhibitor would prevent binding of p27Kip1, resulting in its redistribution to other CDKs. Identification of the critical residues involved in the interaction explains how mutations in Cdk4 and p16INK4a result in loss of kinase inhibition and cancer.     

Expression of mouse c-myb during embryonic development

Three members of the myb gene family, designated as A-myb, B-myb, and c-myb, have been described in many vertebrates. A large body of evidence indicates that the c-myb gene is essential for the development of most hematopoietic lineages. By contrast, the functions of A-myb and B-myb are less well understood. To further explore the relationship between the different myb family members we have compared the expression of A-myb and c-myb during mouse embryogenesis by Northern blotting and by in situ hybridization. In accordance with the important role of c-myb in the hematopoietic system, we detect high levels of c-myb expression in hematopoietic organs such as the fetal liver and the thymus. Surprisingly, we find that high levels of c-myb expression are not restricted to hematopoietic cells. We show that c-myb is strongly expressed in the neural retina and in epithelia of the respiratory tract. The side-by side analysis of c-myb and A-myb expression clearly shows that both genes are expressed in different, but overlapping sets of tissues. Our results suggest that the function of c-myb may not be restricted to hematopoietic cells.     

Expression of second messenger- and cyclin-dependent protein kinases during postnatal development of rat heart

During early postnatal development, cardiomyocytes, which comprise about 80% of ventricular mass and volume, become phenotypically developed to facilitate their contractile functions and terminally differentiated to grow only in size but not in cell number. These changes are due to the expression of contractile proteins as well as the regulation of intracellular signal transduction proteins. In this study, the expression patterns of several protein kinases involved in various cardiac functions and cell-cycle control were analyzed by Western blotting of ventricular extracts from 1-, 10-, 20-, 50-, and 365-day-old rats. The expression level of cAMP-dependent protein kinase was slightly decreased (20%) over the first year, whereas no change was detected in cGMP-dependent protein kinase I. Calmodulin-dependent protein kinase II, which is involved in Ca2+ uptake into the sarcoplasmic reticulum, was increased as much as ten-fold. To the contrary, the expressions of protein kinase C-alpha and iota declined 77% with age. Cyclin-dependent protein kinases (CDKs) such as CDK1, CDK2, CDK4, and CDK5, which are required for cell-cycle progression, abruptly declined to almost undetectable levels after 10-20 days of age. In contrast, other CDK-related kinases, such as CDK8 or Kkialre, did not change significantly or increased up to 50% with age, respectively. Protein kinases implicated in CDK regulation such as CDK7 and Wee1 were either slightly increased in expression or did not change significantly. All of the proteins that were detected in ventricular extracts were also identified in isolated cardiac myocytes in equivalent amounts and analyzed for their relative expression in ten other adult rat tissues.     

Expression of the beta 4 integrin subunit in the mouse heart during embryonic development: retinoic acid advances beta 4 expression

Dye transfer between lens fiber cells and between lens epithelial cells and underlying fiber cells was studied using a wide dynamic range-cooled CCD camera, H2O immersion objectives and image analysis techniques. Each lens was decapsulated by a new technique which leaves the epithelial cells adherent to the lens fiber mass. Lucifer Yellow CH was injected into either single epithelial cells or single fiber cells using the standard whole cell configuration of the patch voltage clamp technique. The results demonstrate extensive dye communication between fiber cells at the lens posterior surface, anterior surface, and equatorial surface. Dye transfer between deep fiber cells was also observed. Dye transfer between approximately 10% of epithelial cells and their underlying fiber cells was apparent when care was taken to yield wide dynamic range images. This was required because the relatively high concentration of dye in the epithelial cell masks the presence of much lower dye concentrations in the underlying fiber cell. A mathematical model which includes dye concentration, time, and spatial spread suggests that those epithelial cells that are coupled to an underlying fiber cell are about as well dye coupled as the epithelial cells themselves. The relatively low dye concentration in a fiber cell is due to its larger volume and diffusion of the dye along the axis of the fiber away from the fiber/epithelial junction.     

Regulation of cyclin-dependent kinase 4 during adipogenesis involves switching of cyclin D subunits and concurrent binding of p18INK4c and p27Kip1

Terminal differentiation of many cell lineages involves an exit from the mitotic cycle and entry into, and maintenance of, a permanent state of G1 arrest. We found that during terminal differentiation of mouse 3T3-L1 preadipocytes, the level of cyclin-dependent kinase 4 (CDK4) remained constant, but the subunit composition of the CDK4 complex underwent a dynamic rearrangement. As 3T3-L1 cells differentiated, the levels of cyclin D1 and cyclin D1-CDK4 complexes declined to negligible levels. Meanwhile, cyclins D2 and D3 levels and their associations with CDK4 increased transiently and persistently, respectively, with cyclin D3 becoming the predominant cyclin partner of CDK4 in mature adipocytes. At least five CDK inhibitors are expressed during the differentiation program of 3T3-L1 cells. Both p15INK4b and p16INK4a continuously declined to undetectable levels immediately after differentiation induction. p21 was transiently expressed during the exit of 3T3-L1 cells from mitotic clonal expansion and then decreased to undetectable levels in mature adipocytes. The level of p27KiP1 and p27-CDK4 complexes remain high during differentiation and in mature adipocytes. Distinctly, there is a remarkable induction of p18INK4c mRNA and protein that was not seen in the closely related nondifferentiating 3T3-C2 cell line, suggesting that p18 induction in 3T3-L1 cells is related to cell differentiation, not cell cycle arrest. The pRb kinase activity of cyclin D3 and CDK4 was not detected in quiescent 3T3-L1 cells and was then induced as the cells entered the mitotic clonal expansion phase. Unexpectedly, cyclin D3 and CDK4 pRb kinase activity remained high after 3T3-L1 cells completed their mitotic division and was still readily detectable in mature adipocytes. Our study reveals an active regulation, rather than passive inhibition, of CDK4 activity during adipocyte differentiation. Two central features of this complex regulation are switching of activating cyclin D subunits and concurrent binding by the p18 and p27 CDK inhibitors.     

Requirement of cyclin E-Cdk2 inhibition in p16(INK4a)-mediated growth suppression

Loss-of-function mutations of p16(INK4a) have been identified in a large number of human tumors. An established biochemical function of p16 is its ability to specifically inhibit cyclin D-dependent kinases in vitro, and this inhibition is believed to be the cause of the p16-mediated G1 cell cycle arrest after reintroduction of p16 into p16-deficient tumor cells. However, a mutant of Cdk4, Cdk4(N158), designed to specifically inhibit cyclin D-dependent kinases through dominant negative interference, was unable to arrest the cell cycle of the same cells (S. van den Heuvel and E. Harlow, Science 262:2050-2054, 1993). In this study, we determined functional differences between p16 and Cdk4(N158). We show that p16 and Cdk4(N158) inhibit the kinase activity of cellular cyclin D1 complexes through different mechanisms. p16 dissociated cyclin D1-Cdk4 complexes with the release of bound p27(KIP1), while Cdk4(N158) formed complexes with cyclin D1 and p27. In cells induced to overexpress p16, a higher portion of cellular p27 formed complexes with cyclin E-Cdk2, and Cdk2-associated kinase activities were correspondingly inhibited. Cells engineered to express moderately elevated levels of cyclin E became resistant to p16-mediated growth suppression. These results demonstrate that inhibition of cyclin D-dependent kinase activity may not be sufficient to cause G1 arrest in actively proliferating tumor cells. Inhibition of cyclin E-dependent kinases is required in p16-mediated growth suppression.     

Expression of laminin and nidogen genes during the postimplantation development of the mouse placenta

The expression patterns of laminin A, B1, B2, and nidogen genes were identified by in situ hybridization in postimplantation mouse extraembryonic tissues and maternal decidua during the period when the chorioallantoic placenta is established. Laminin and nidogen genes were not coordinately expressed either in the decidua or in trophoblast cells, indicating that these genes are regulated independently in these cell types during the establishment of the placenta. Laminin A mRNA was absent from the decidua except in the outer layer of cells adjacent to the myometrium and in the central decidual zone adjacent to the remnant of the uterine epithelium on Day 9. At this stage laminin B1, B2, and nidogen genes were strongly expressed in these cells and also in other regions of the decidua. Laminin B1 mRNA was present at higher levels in the decidua capsularis than in the decidua basalis, while nidogen mRNA showed highest expression in the decidua basalis. Laminin B2 mRNA was produced uniformly throughout the decidua at very high levels, suggesting that laminin B2 chains may be an important component of the decidual matrix. By Day 11, the nidogen gene was expressed only in endothelial cells lining the maternal blood spaces within the decidua. Laminin B1 and nidogen mRNAs were found at high levels within trophoblast giant cells at all stages, while laminin A mRNA was detected in trophoblast giant cells at later stages and laminin B2 mRNA was not produced in high levels by these cells. The patterns of gene expression show a very high degree of regional specialization, suggesting that the extracellular matrices in different regions of the decidua and extraembryonic membranes are likely to be composed of quite different ratios of laminin and nidogen polypeptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuronal expression of cycline dependent kinase inhibitors of the INK4 family in Alzheimer's disease

Neurodegeneration and cell death in Alzheimer's disease might be associated with aberrant proliferative mechanisms and activation of cell-cycle related events. We reported previously on the elevated expression of the cyclin dependent kinase inhibitor p16INK4a in Alzheimer's disease closely associated with neurofibrillary degeneration. In the present study, we demonstrate that other members of the INK4-family of cyclin dependent kinase inhibitors such as p15INK4b, p18INK4c and p19INK4d that bind directly to cdk4/6 or to complexes of cdk4/6 with D-type cyclins are all elevated. In contrast, no indication of altered expression of the cyclin dependent kinase inhibitors p21Cip1 and p27Kip1 were observed. Inhibitors of the INK4-family were strongly expressed in tangle-bearing neurones and neuritic components of plaques. A much lower expression was also seen in astrocytes. These findings add further evidence to the suggestion that a dysfunction of cell cycle regulation is of critical importance in the pathomechanism of Alzheimer's disease.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the mouse uterus during the peri-implantation period

In this study the SEB-activated LAK cytotoxicity was identified and characterized in human peripheral blood lymphocytes (PBMC). After 3 days of SEB stimulation, the PBMC acquired a cytotoxicity against traditional LAK targets, K-562 and Daudi, beside that human glomerular endothelial cells (HGEC) were effectively lysed. The magnetic separation of SEB-stimulated CD5+ T cells revealed that the dominant LAK cytotoxicity remained in the CD5- lymphocyte fraction. The major part of the SEB-generated cytotoxicity of CD5- cells could be blocked with specific antibodies to IL-2 and IFN-gamma. The IFN-gamma pretreatment of HGEC reduced the target sensitivity, but because of the upregulation of MHC class II on HGEC surface, these cells were able to present SEB to CD5+ cells. These results suggested that in bacterial superantigen-mediated infection, the non-T (NK cells-derived) LAK cells might have a primary pathogenic role, and the adverse effect of IFN-gamma, that was massively secreted from superantigen-stimulated cells, requires greater consideration.     

Expression of fas antigen in the normal mouse brain

In this paper, the first of a series dealing with the development of a methodology for assessing quality of ambulatory care, a sample of 270 outpatients from the same health center were presented with a list of 12 questions. Although different versions of the questionnaire were tested, we found a high degree of agreement between the results. The findings indicate that the parameter "satisfaction" lends itself readily to measurement, thus becoming a useful instrument for guiding active intervention.     

Expression of copper trafficking genes in the mouse brain

Copper homeostasis in the brain must be strictly maintained, since copper is an essential trace element and is potentially toxic. To understand the mechanism of copper homeostasis in the brain, we cloned several mouse homologues of copper trafficking genes and performed in situ hybridization histochemistry. mCTR1, mATX1, and mATP7a were highly expressed in the choroid plexus, indicating that the choroid plexus uses the trafficking pathway from uptake to efflux to transport copper to the cerebrospinal fluids. We suggest that these genes may regulate copper concentration in the brain through the choroid plexus.     

Role of the mitogen-activated protein kinases and tyrosine kinases during leukotriene B4-induced eosinophil activation

Exposure of guinea-pig eosinophils to leukotriene B4 (LTB4; 1 microM) resulted in a rapid generation of H2O2 (index of NADPH oxidase activation), stimulated [3H]arachidonic acid (AA) release (index of phospholipase A2 activity), and promoted CD18-dependent homotypic aggregation. Under similar conditions, LTB4 (1 microM) induced a rapid activation of extracellular-regulated kinases-1 and 2 (ERK-1/2) but not c-jun N-terminal kinases 46 and 54 (JNK-46/54) or p38 mitogen-activated protein kinase (p38 MAP kinase). To examine the role of ERK-1/2 in the mechanism of eosinophil activation, a selective inhibitor of MAP kinase kinase-1/2 (MEK-1/2), PD098059, was employed. However, PD 098059 at concentrations that attenuated ERK-1/2 activation had no significant affect on eosinophil activation. In contrast, a role for tyrosine kinases in LTB4-induced eosinophil activation was suggested by studies with the tyrosine kinase inhibitors, herbimycin A and lavendustin A. However, the results of those experiments implied divergent pathways for the control of eosinophil responses because the inhibitors were more effective at attenuating H2O2 generation than [3H]AA release, and had little effect on homotypic aggregation.     

Genetic and epigenetic alterations of the cyclin-dependent kinase inhibitors p15INK4b and p16INK4a in human thyroid carcinoma cell lines and primary thyroid carcinomas

BACKGROUND: D-type cyclins, in association with the cyclin-dependent kinases CDK4 and CDK6, promote progression through the G1 phase of the cell cycle. CDK activity is modulated by inhibitors such as p15INK4b and p16INK4a. Loss of function of p15INK4b and p16INK4a (multiple tumor suppressor-I and CDK4 inhibitor) determines impairment in the control of the cell cycle and contributes to the transformation of several cell types. METHODS: The authors examined 20 thyroid neoplasms (12 papillary carcinomas and 8 follicular adenomas) and 4 human thyroid carcinoma cell lines for gene mutations and epigenetic modifications of the p15INK4b and p16INK4a genes by Southern blot analysis, single strand conformation polymorphism, and a polymerase chain reaction-based methylation assay. RESULTS: Abnormalities of p16 were found in the four cell lines studied. In follicular carcinoma (WRO) cells, both the p15 and p16 genes were homozygously deleted. Undifferentiated carcinoma (FRO) cells had a nonsense point mutation at codon 72 (CGA-TGA, Arg-Stop) of p16, whereas the poorly differentiated papillary carcinoma (NPA) line harbored a point mutation at the exon 1-intron 1 boundary that altered the donor splicing site and caused an aberrantly spliced form of p16INK4a. Furthermore, p16 allelic loss was evident in the DNA of both FRO and NPA cells. Finally, p16 expression was absent in the ARO cell line, likely due to a de novo methylation of exon 1 of p16INK4a. Regarding the primary thyroid tumors, a missense point mutation at codon 91 was found in 1 of 12 papillary thyroid carcinomas (GCC-GTC, Ala-Val). No mutations were found in follicular adenomas. However, in 6 of 20 primary tumors there was hypermethylation at exon 1 of p16. CONCLUSIONS: The high prevalence of p15 and p16 mutations in the cell lines described suggests involvement of these genes in immortalization in vitro. The p16 defects may have preexisted in a small subclone of the primary tumor that were selected for in vitro. Alternatively, p16 mutations may have arisen de novo during cell culture. Mutations of p15INK4b and p16INK4a do not appear to be critical events in the development of follicular adenomas or papillary carcinomas. However, de novo methylation of the 5' CpG island of p16 is common in primary tumors, indicating that the function of this gene may be lost as an epigenetic event during disease progression.     

Inactivation of the p15INK4B and p16INK4 genes in hematologic malignancies

The recently discovered p15INK4B and p16INK4 genes encoding cell cycle regulating proteins, map to a region on chromosome 9p21 that is commonly deleted in a variety of malignant diseases. The p16INK4 gene has now been shown to be a tumor suppressor gene. It is frequently inactivated in cancer and is possibly the second most often mutated gene in human malignant disease after p53. The role of the p15INK4B and p16INK4 genes in hematologic malignancies has been the subject of intense investigation since their discovery. In this review we address the function and possible role in tumorigenesis of the p15INK4B and p16INK4 genes and discuss their significance as prognostic markers in hematologic malignancies.