ATP hydrolysis catalyzed by human replication factor C requires participation of multiple subunits

Human replication factor C (hRFC) is a five-subunit protein complex (p140, p40, p38, p37, and p36) that acts to catalytically load proliferating cell nuclear antigen onto DNA, where it recruits DNA polymerase delta or epsilon to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomplex of hRFC consisting of three subunits (p40, p37, and p36) contained DNA-dependent ATPase activity. However, it is not clear which subunit(s) hydrolyzes ATP, as all five subunits include potential ATP binding sites. In this report, we introduced point mutations in the putative ATP-binding sequences of each hRFC subunit and examined the properties of the resulting mutant hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduced replication activity of the hRFC complex and the ATPase activity of the hRFC or the p40.p37.p36 complex. A mutation in the ATP binding site of the p38 subunit did not alter the replication activity of hRFC. These findings indicate that the replication activity of hRFC is dependent on efficient ATP hydrolysis contributed to by the action of four hRFC subunits.     

Identification of specific phosphoproteins in nuclease-digested chromatin subunits

The angiocardiographic, echocardiographic, and, where available, the necropsy findings were correlated in 32 cases of primitive ventricle. Single probe echocardiography was shown to be a reliable and accurate technique for diagnosis of primitive ventricle; the ventricular and atrioventricular valve appearances were characteristic, and the outlet chamber was usually recognised when present, though it was not possible to say whether it was rigt or left sided. Abnormalities of the atrioventricular valves were more accurately shown by echocardiography than by angiocardiography though the two techniques were shown to be complementary in the overall diagnostic process.     

Identification of multiple human calcitonin receptor isoforms: heterologous expression and pharmacological characterization

The human breast carcinoma cell line T47D is known to express high-affinity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA from T47D mRNA resulted in the identification of two different cDNAs that encode distinct receptor isoforms, h alpha CTR and h beta CTR. The two cDNAs are identical except that the h alpha CTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first cytosolic loop of the receptor. Stable transfection of each receptor cDNA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin (h alpha CTR Kd 0.09 nM, h beta CTR Kd 0.12 nM). Ligand competition binding studies did not reveal any significant pharmacological difference between the receptor isoforms. In transfected MEL cells and COS-1 cells the h beta CTR isoform was expressed at tenfold higher levels than the h alpha CTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in beta-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.     

Simultaneous analysis of cyclin and oncogene expression using multiple monoclonal antibody immunoblots

Cell dysfunction or dysregulation in cancer generally results from complex gene interactions, numerous cellular events and environmental influences which modify gene expression or post-translational protein modifications. Genetic analysis in itself cannot always predict or diagnose multigenic diseases. The major technical difficulty is thus to detect, identify and measure simultaneously the expression of several genes and the post-translational modifications of their products. In order to progress to this direction, this paper describes a simple immunoblot method using several monoclonal anti-bodies to simultaneously analyze oncogene expression and cell cycle specific checkpoints in patient solid biopsies and transformed cell lines. One mg of normal human liver biopsy and HEPG2 (hepatoblastoma-derived cell line) protein samples have been separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were stained with amido black, scanned and tested separately with the nine monoclonal antibodies p53, c-myc, PCNA, MEK1, pan-ras, Cip1, Cdc2, Kip1, and TCTP. The nine antibodies of interest were then combined to form a mixture, and simultaneously used as the primary antibodies. This antibody mixture simultaneously detected the nine proteins of interest on both samples and it demonstrated the extensive expression changes and the presence of various isoforms most likely due to post-translational modifications of gene products.     

Identification of two minor subunits in the pilus of Haemophilus influenzae

Haemophilus influenzae type b (Hib) organisms produce pili, which mediate attachment to human cells and are multimeric structures composed of a 24-kDa subunit called pilin or HifA. Although pili from other organisms contain additional proteins accessory to pilin, no structural components other than pilin have been identified in Hib pili. Previous analysis of a Hib pilus gene cluster, however, suggested that two genes, hifD and hifE, may encode additional pilus subunits. To determine if hifD and hifE encode pilus components, the genes were overexpressed in Escherichia coli and the resulting proteins were purified and used to raise polyclonal antisera. Antisera raised against C-terminal HifD and HifE fragments reacted with H. influenzae HifD and HifE proteins, respectively, on Western immunoblots. Western immunoblot analysis of immunoprecipitated Hib pili demonstrated that HifD and HifE copurified with pili. In enzyme-linked immunosorbent assays, antisera raised against a recombinant HifE protein that contained most of the mature protein reacted more to piliated Hib than to nonpiliated Hib or to a mutant containing a hifE gene insertion. Immunoelectron microscopy confirmed that the HifE antiserum bound to pili and demonstrated that the antiserum bound predominantly to the pilus tips. These data indicate that HifD and HifE are pilus subunits. Adherence inhibition studies demonstrated that the HifE antiserum completely blocked pilus-mediated hemagglutination, suggesting that HifE mediates pilus adherence.     

Enhanced expression of cyclin D1 in senescent human fibroblasts

When human fibroblast, TIG-1, was growth-stimulated with fetal bovine serum, the induction level of cell cycle-dependent genes was generally much lower in senescent cells than in young counterparts. Exceptionally, the expression level of cyclin D1 in senescent cells was constitutively higher than in young cells and further increased after serum stimulation, which was confirmed by Northern and Western blots and immunoprecipitation. This was also true in other human diploid fibroblast lines, TIG-3 and MRC-5. However, cyclin D1-dependent kinase activity was not detected in senescent cells. When sense- or antisense-cyclin D1 cDNA driven by beta-actin promoter was transfected into young TIG-1 cells, the number of appeared colonies from sense-strand transfected cultures was lower than that from antisense-strand-transfected ones. However, clones expressing cyclin D1 at low or undetectable level which were isolated after transfection with antisense-cyclin D1 proliferated up to the same division limit as untransfected and sense-strand transfected cells. Four clones of SV40-transformed TIG-1 expressed cyclin D1 at moderate levels during their extended proliferative lifespan. It appears that, if the extremely overexpressed cyclin D1 could cause an inhibition of cell proliferation at senescent stage, cellular senescence occurs regardless of overexpression of cyclin D1.     

Role of cyclin E and cyclin E-dependent kinase in mitogenic stimulation by cementum-derived growth factor in human fibroblasts

Cementum-derived growth factor (CGF) is a 14 kDa polypeptide sequestered in tooth cementum. It is an IGF-I like molecule that is weakly mitogenic to fibroblasts, but its mitogenic action is synergistically potentiated in the presence of epidermal growth factor (EGF) or serum. We have examined whether the CGF affects cyclin E levels and the activity of cyclin-dependent kinase (Cdk) associated with this cyclin, and whether these changes contribute to the synergism in mitogenic activity between CGF and EGF. Optimal DNA synthesis by serum-starved human gingival fibroblasts required the presence of CGF for 0-12 h and EGF for 0-3 h. Therefore, cells were serum starved for 48 h and then exposed to CGF, EGF, or CGF + EGF. Cells incubated with 10% fetal bovine serum (FBS) served as positive controls. At various time points after the addition of growth factors, cyclin E levels were examined by Western analysis. Cdk associated with cyclin E was immunoprecipitated with anti-cyclin E antibody and kinase activity was measured using H1 histone as substrate. Cyclin E and the H1 kinase activity levels increased after 8-12 h in cells exposed to CGF and in positive controls exposed to 10% FBS. They returned to basal level 4 h later in cells exposed to CGF alone, whereas in the presence of CGF + EGF and FBS they remained elevated for up to 20 h. The cyclin E levels did not increase in the presence of EGF alone. Cyclin-dependent kinase inhibitors p21cip1 and p27kip1 were barely detectable in these cells. Fibroblasts transfected with LXSN-cyclin E, a retroviral vector containing cyclin E cDNA, overexpressed cyclin E and their steady-state cyclin E-Cdk activity was higher than control cells. DNA synthesis by cyclin E overexpressing cells was higher, but optimal DNA synthesis by these cells required the presence of CGF and EGF. These results show that CGF action involves an increase in the levels of cyclin E and E-Cdk activity and that the higher levels are maintained in the presence of both CGF and EGF. They also indicate that sustained high cyclin E levels and Cdk2 activity during G1 phase are necessary, but not sufficient, for optimal mitogenic response in human fibroblasts.     

Identification of a p130 domain mediating interactions with cyclin A/cdk 2 and cyclin E/cdk 2 complexes

P130 shares structural and functional homology with pRb and p107. One property common to p107 and p130, but not to pRb, is the ability to stably interact with cyclin A/cdk2 and cyclin E/cdk2 complexes in vitro and in vivo. Using GST-p130 fusion proteins representing various regions of p130, baculovirus-produced cyclin A/cdk2 and cyclin E/cdk2 complexes were found to interact with residues within a part of p130 known as the spacer region. Cyclin E was able to bind the p130 spacer region in the presence or absence of cdk2 whereas cyclin A binding was dependent upon the presence of cdk2. The smallest p130 fusion protein sufficient to interact with cyclin A/cdk2 or cyclin E/cdk2 complexes contained p130 amino acids 652-698 and deletion of p130 amino acids 680-682 abolished binding to both of the cyclin/cdk2 complexes. When overexpressed in C33A cells, a p130 mutant containing a deletion of amino acids 620-697 was unable to form complexes with either cyclin A or cyclin E. This p130 mutant was at least as active as wild type p130 in suppressing the growth of G418 resistant colonies when overexpressed in C33A or SAOS-2 cells.     

Interactions between the human RNA polymerase II subunits

As an initial approach to characterizing the molecular structure of the human RNA polymerase II (hRPB), we systematically investigated the protein-protein contacts that the subunits of this enzyme may establish with each other. To this end, we applied a glutathione S-transferase-pulldown assay to extracts from Sf9 insect cells, which were coinfected with all possible combinations of recombinant baculoviruses expressing hRPB subunits, either as untagged polypeptides or as glutathione S-transferase fusion proteins. This is the first comprehensive study of interactions between eukaryotic RNA polymerase subunits; among the 116 combinations of hRPB subunits tested, 56 showed significant to strong interactions, whereas 60 were negative. Within the intricate network of interactions, subunits hRPB3 and hRPB5 play a central role in polymerase organization. These subunits, which are able to homodimerize and to interact, may constitute the nucleation center for polymerase assembly, by providing a large interface to most of the other subunits.     

Interactions between subunits of the human epithelial sodium channel

The human epithelial sodium channel (hENaC) mediates Na+ transport across the apical membrane of epithelia, and mutations in hENaC result in hypertensive and salt-wasting diseases. In heterologous expression systems, maximal hENaC function requires co-expression of three homologous proteins, the alpha, beta, and gammahENaC subunits, suggesting that hENaC subunits interact to form a multimeric channel complex. Using a co-immunoprecipitation assay, we found that hENaC subunits associated tightly to form homo- and heteromeric complexes and that the association between subunits occurred early in channel biosynthesis. Deletion analysis of gammahENaC revealed that the N terminus was sufficient but not necessary for co-precipitation of alphahENaC, and that both the N terminus and the second transmembrane segment (M2) were required for gamma subunit function. The biochemical studies were supported by functional studies. Co-expression of gamma subunits lacking M2 with full-length hENaC subunits revealed an inhibitory effect on hENaC channel function that appeared to be mediated by the cytoplasmic N terminus of gamma, and was consistent with the assembly of nonfunctional subunits into the channel complex. We conclude that the N terminus of gammahENaC is involved in channel assembly.     

Cell cycle-related variation and tissue-restricted expression of human cyclin D1 protein

Recent evidence from genetic studies suggests that abnormalities of some of the members of the cyclin superfamily may be intimately associated with tumourigenesis, most likely through deregulation of the cell cycle control. In an attempt to elucidate the potential role of cyclin D1 (a gene located within the 11q13 amplicon and a candidate BCL-1, PRAD-1 oncogene) in the pathogenesis of human neoplasias, we have developed and characterized a novel monoclonal antibody specifically recognizing cyclin D1 protein in various assays including immunohistochemistry on frozen and paraffin sections. Using the DCS-6 antibody as a tool, we now show a characteristic cell cycle-dependent variation of the cyclin D1 protein in human cultured cells and report on the first immunohistochemical study of this G1 cyclin in a range of normal human tissues and breast carcinomas. Analysis of normal tissues revealed generally low levels of cyclin D1 protein, mainly restricted to the proliferative zones of some epithelial tissues, and the lack of its expression in several human tissues including lymph nodes, spleen, and tonsils. In contrast, pronounced overexpression/nuclear accumulation of cyclin D1 was found in 37 per cent of cases in a series of 35 primary ductal carcinomas of the breast. We conclude that the DCS-6 antibody provides a potentially useful tool for the establishment of simple methods suitable for verifying any diagnostic and/or prognostic value of this novel marker on large series of histological specimens and opens the way for biochemical, immunocytochemical, and immunohistochemical studies of the role played by cyclin D1 aberrations in human oncogenesis.     

The expression of voltage-dependent calcium channel beta subunits in human cerebellum

The beta subunits of voltage-dependent calcium channels, exert marked regulatory effects on the biophysical and pharmacological properties of this diverse group of ion channels. However, little is known about the comparative neuronal expression of the four classes of beta genes in the CNS. In the current investigation we have closely mapped the distribution of beta1, beta2, beta3 and beta4 subunits in the human cerebellum by both in situ messenger RNA hybridization and protein immunohistochemistry. To our knowledge, these studies represent the first experiments in any species in which the detailed localization of each beta protein has been comparatively mapped in a neuroanatomically-based investigation. The data indicate that all four classes of beta subunits are found in the cerebellum and suggest that in certain neuronal populations they may each be expressed within the same cell. Novel immunohistochemical results further exemplify that the beta voltage-dependent calcium channel subunits are regionally distributed in a highly specific manner and studies of Purkinje cells indicate that this may occur at the subcellular level. Preliminary indication of the subunit composition of certain native voltage-dependent calcium channels is suggested by the observation that the distribution of the beta3 subunit in the cerebellar cortex is identical to that of alpha(1E). Our cumulative data are consistent with the emerging view that different native alpha1/beta subunit associations occur in the CNS.     

Analysis of chaperonin-containing TCP-1 subunits in the human keratinocyte two-dimensional protein database: further characterisation of antibodies to individual subunits

The chaperonin-containing TCP-1 (CCT), found in the eukaryotic cytosol, is currently the focus of extensive research. CCT consists of at least eight different subunit types encoded by independent but related genes, and a set of antibodies that recognise individual subunits has proved useful in the characterisation and functional analysis of CCT. These antibodies were used to identify subunits of CCT in the human keratinocyte two-dimensional protein database. Accurate values for the pI and molecular mass of human CCT subunits were determined from the database, and biological data was obtained regarding changes in subunit levels in response to extracellular agents and growth conditions. The second part of the study describes the characterisation of seven monoclonal antibodies raised against mouse TCP-1, also known as CCT alpha, using a combination of epitope mapping and immunoblot analysis of protein extracts from different species and tissue types. Some antibodies were not monospecific for TCP-1, and a number of epitope-related proteins were identified.     

Increased expression of cyclin D2 during multiple states of growth arrest in primary and established cells

Cyclin D2 is a member of the family of D-type cyclins that is implicated in cell cycle regulation, differentiation, and oncogenic transformation. To better understand the role of this cyclin in the control of cell proliferation, cyclin D2 expression was monitored under various growth conditions in primary human and established murine fibroblasts. In different states of cellular growth arrest initiated by contact inhibition, serum starvation, or cellular senescence, marked increases (5- to 20-fold) were seen in the expression levels of cyclin D2 mRNA and protein. Indirect immunofluorescence studies showed that cyclin D2 protein localized to the nucleus in G0, suggesting a nuclear function for cyclin D2 in quiescent cells. Cyclin D2 was also found to be associated with the cyclin-dependent kinases CDK2 and CDK4 but not CDK6 during growth arrest. Cyclin D2-CDK2 complexes increased in amounts but were inactive as histone H1 kinases in quiescent cells. Transient transfection and needle microinjection of cyclin D2 expression constructs demonstrated that overexpression of cyclin D2 protein efficiently inhibited cell cycle progression and DNA synthesis. These data suggest that in addition to a role in promoting cell cycle progression through phosphorylation of retinoblastoma family proteins in some cell systems, cyclin D2 may contribute to the induction and/or maintenance of a nonproliferative state, possibly through sequestration of the CDK2 catalytic subunit.