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Biomedical subjects

F L Hall

Publications and source records attributed to F L Hall.

At least 37 records · Page 2Linked to original sources
PubMed1997

Targeting retroviral vectors to vascular lesions by genetic engineering of the MoMLV gp70 envelope protein.

Targeted gene delivery to vascular lesions is a major challenge in the development of gene therapy protocols for cardiovascular diseases. One approach would be to enable retroviral vectors to accumulate at sites of vascular injury and enhance local vector concentration. An early step in wound repair is the adhesion of platelets to collagen exposed from damaged vasculature. Hence, the Moloney murine leukemia virus (MoMLV) envelope (env) protein was engineered to incorporate a high-affinity collagen-binding domain derived from von Willebrand clotting factor, and expressed in Escherichia coli and in mammalian cells. The chimeric env protein bound tightly to collagen, and virions bearing this collagen-binding env protein exhibited viral titers approaching those of virions expressing wild-type (WT) env protein. The chimeric virions were concentrated on collagen matrices, and they retained their infectivity under conditions in which virions bearing WT env protein were washed away. Targeted delivery of the chimeric env protein to injured mouse aorta and selective binding of the collagen-targeted virions to injured rabbit artery were observed. In comparative studies, vascular smooth muscle cell transduction was demonstrated in catheter-injured carotid arteries following infusion of collagen-targeted virions but not of virions bearing WT env protein. Taken together, these observations demonstrate the ability of collagen-targeted virions to localize gene delivery to sites of vascular injury.

3T3 Cells↗
PubMed1997

Retroviral vector-mediated transfer of an antisense cyclin G1 construct inhibits osteosarcoma tumor growth in nude mice.

Metastatic osteosarcoma is a potential target for gene therapy, because conventional therapies are only palliative and metastatic disease is invariably fatal. Overexpression of the cyclin G1 (CYCG1) gene is frequently observed in human osteosarcoma cells, and its continued expression is found to be essential for their survival. Previously, we reported that down-regulation of cyclin G1 protein expression induced cytostatic and cytocidal effects in human MG-63 osteosarcoma cells (Skotzko et al., Cancer Research, 1995). Here, we extend these findings in a tumorigenic MNNG/HOS cell line and report on the effective inhibition of tumor growth in vivo by an antisense cyclin G1 retroviral vector when delivered as concentrated high titer vector supernatants directly into rapidly growing subcutaneous tumors in athymic nude mice. Histologic sections from the antisense cyclin G1 vector-treated tumors showed decreased mitotic indices and increased stroma formation within the residual tumors. Furthermore, in situ analysis of the cell-cycle kinetics of residual tumor cells revealed a decrease in the number of cells in S and G2/M phases of the cell cycle concomittant with an accumulation of cells in the G1 phase. Taken together, these studies demonstrate in vivo efficacy of a high-titer antisense cyclin G1 retroviral vector in an animal model of osteosarcoma.

Animals↗
PubMed1997

Capture and expansion of bone marrow-derived mesenchymal progenitor cells with a transforming growth factor-beta1-von Willebrand's factor fusion protein for retrovirus-mediated delivery of coagulation factor IX.

Mesenchymal stem cells give rise to the progenitors of many differentiated phenotypes, including osteocytes, chrondocytes, myocytes, adipocytes, fibroblasts, and marrow stromal cells, which are capable of self-renewal and undergo expansion in the presence of transforming growth factor-beta1 (TGF-beta1). The present study was designed to test the concept that mesenchymal progenitor cells could be selected and expanded by virtue of their intrinsic physiologic responses to TGF-beta1. Human bone marrow aspirates were initially cultured, under low serum conditions, in collagen pads or gels impregnated with a genetically engineered TGF-beta1 fusion protein bearing an auxiliary von Willebrand's factor-derived collagen-binding domain (TGF-beta1-vWF). Histologic examination of TGF-beta1-vWF-supplemented collagen pads from 8-day cultures revealed the selective survival of a population of mononuclear blastoid cells. The TGF-beta-responsive cells were expanded to form stromal/fibroblastic colonies by serum reconstitution, and further to form osteogenic colonies upon supplementation with osteoinductive factors. In comparative studies, both marrow-derived progenitor cells and mature stromal cells were transduced with a retroviral vector bearing a human factor IX construct. Both the transduced progenitor cells and mature stromal cells expressed the factor IX transgene at levels comparable to those reported for human fibroblasts. Transplantation of murine progenitor cells bearing the human factor IX vector into syngeneic B6CBA mice resulted in detectable circulating levels of the human factor IX antigen. Taken together, these data demonstrate a novel physiologic approach for the selection of mesenchymal precursor cells followed by mitotic expansion, transduction, and transplantation of these progenitor cells with retroviral vectors bearing therapeutic genes.

Animals↗
PubMed1997

Downregulation of cyclin G1 expression by retrovirus-mediated antisense gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation.

BACKGROUND: The contemporary treatment of coronary athero-occlusive disease by percutaneous transluminal coronary angioplasty is hampered by maladaptive wound healing, resulting in significant failure rates. Morbid sequelae include smooth muscle cell (SMC) hyperplasia and restenosis due to vascular neointima formation. METHODS AND RESULTS: In this study, we examined the inhibitory effects of a concentrated retroviral vector bearing an antisense cyclin G1 gene on aortic SMC proliferation in vitro and on neointima formation in vivo in a rat carotid injury model of restenosis. Retroviral vectors bearing an antisense cyclin G1 construct inhibited the proliferation of transduced aortic SMCs in 2- to 6-day cultures, concomitant with down-regulation of cyclin G1 protein expression and decreased [3H]thymidine incorporation into DNA. Morphological examination showed evidence of cytolysis, giant syncytia formation, and apoptotic changes evidenced by overt cell shrinkage, nuclear fragmentation, and specific immunostaining of nascent 3'-OH DNA ends generated by endonuclease-mediated DNA fragmentation. Pronounced "bystander effects" including neighboring cells were noted in aortic SMCs transduced with the antisense cyclin G1 vector, as determined by quantitative assays and fluorescent labeling of nontransduced cells. In an in vitro tissue injury model, the proliferation and migration of antisense cyclin G1 vector-transduced aortic SMCs were inhibited. Moreover, in vivo delivery of high-titer antisense cyclin G1 vector supernatant to the balloon-injured rat carotid artery in vivo resulted in a significant reduction in neointima formation. CONCLUSIONS: These findings represent the first demonstration of the inhibitory effects of an antisense cyclin G1 retroviral vector on nonneoplastic cell proliferation. Taken together, these data affirm the potential utility of antisense cyclin G1 constructs in the development of novel gene therapy approaches to vascular restenosis.

Animals↗
PubMed1997

Refolding of a recombinant collagen-targeted TGF-beta2 fusion protein expressed in Escherichia coli.

In this study, a tripartite transforming growth factor-beta (TGF-beta2) fusion protein bearing an N-terminal purification tag and an auxiliary collagen binding decapeptide has been constructed and expressed at high levels in Escherichia coli. The resulting recombinant protein accumulates in an insoluble and biologically inactive inclusion-body complex. The insoluble protein was solubilized in guanidine hydrochloride and a Ni-chelating affinity column was utilized to isolate the 13.5-kDa TGF-beta2 fusion protein, which was then refolded into its native conformation under controlled redox conditions. The formation of native homodimers was monitored by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gels and the bioactivity determined by a quantitative TGF-beta assay system using mink lung epithelial cells transfected with a plasminogen activator inhibitor-1 promoter/luciferase reporter plasmid. To optimize yields, renaturation conditions including denaturants, limiting protein concentrations, redox ratios, dialysis conditions, and refolding kinetics were studied and monitored by bioactivity. These studies demonstrate that recombinant TGF-beta2 fusion proteins can be produced in E. coli and renatured into biologically active homodimers. Furthermore, they confirm that the auxiliary collagen binding domain effectively targets the recombinant growth factor to type I collagen. Taken together, these studies advance the technology necessary to generate large quantities of targeted TGF-beta fusion proteins for specific biomedical applications.

Biological Assay↗
PubMed1997

Cyclin D1 antisense RNA destabilizes pRb and retards lung cancer cell growth.

To investigate the role of cyclin D1 in the regulation of lung cancer cell growth, we created five stably transfected cell lines carrying a cyclin D1 antisense construct. The transfected cells exhibited a marked decrease in the rate of cell growth, in contrast to the original lines (A549 and NCI-H441). The expression of several cell cycle-regulating proteins, including cyclin A, the cyclin-dependent kinases (cdk) 2 and cdk4, in addition to cyclin D1 itself, was markedly decreased. The expression of one cdk inhibitor, p21WAF1/CIP1, increased in the A549-derived cell lines. A specific target of cyclin D1 activity, the growth-suppressing product of the retinoblastoma gene, pRb, exhibited decreased expression and a decreased level of phosphorylation in the transfected cells. Decreased expression of pRb due to a significant increase in its turnover rate suggested that the stability of the protein may depend on phosphorylation by cyclin D1-dependent cdk activity. In addition to the impact on pRb stability, decreased expression of cyclin D1 induced susceptibility to cell death after withdrawal of exogenous growth factors in the antisense transfected cell lines, a response that was not observed in the original cancer cell lines. We conclude that abrogation of cyclin D1 overexpression in lung cancer cells disrupts several key pathways that are required for uncontrolled cell growth and induces those that lead to cell death after growth factor deprivation. Therefore, we speculate that use of antisense cyclin D1 expression in appropriate gene vectors could be a useful method for retarding lung cancer cell growth in accessible tumors such as those of the lung epithelium.

CDC2-CDC28 Kinases↗
PubMed1997

WAF1/Cip1 gene polymorphism and expression in carcinomas of the breast, ovary, and endometrium.

The p53 gene is altered in approximately 50% of human cancers and is considered to be important in the pathogenesis of these malignancies. The p53 protein product regulates the transition from G1 to S phase of the cell cycle and entry to the DNA damage repair pathway. As alterations in this pathway appear to be important in a variety of human cancers, downstream effector proteins of p53 are potential sites for somatic alterations. WAF1/Cip1, also known as WAF1, Cip1, sdi1, or CAP20, codes for a 21-kd protein (p21WAF1/Cip1), which was recently described as a universal inhibitor of cyclins and is thus critical in cell cycle control. Mutations in WAF1/Cip1 are potentially important in human malignancies because they could affect the control of the cell cycle. To understand whether mutations of WAF1/Cip1 occur in cancer, we screened 53 cases of invasive breast carcinoma, 35 cases of ductal carcinoma in situ (DCIS), 53 ovarian carcinomas, and 47 endometrial carcinomas in the second exon of WAF1/Cip1 (90% of the open reading frame). p21WAF1/Cip1 expression was characterized with immunohistochemistry. Cells from the blood of 21 normal individuals were also characterized using single-strand conformational polymorphism analysis, DNA sequencing and restriction analysis. Single-strand conformational polymorphism analysis demonstrated an altered mobility pattern for exon 2 in 12 invasive breast cancers (22.6%), 5 DCIS of the breast (14%), 8 invasive ovarian carcinomas (15%), and 9 endometrial carcinomas (19%). In total, 209 samples were screened, and 38 cases (18.2%) had an altered codon 31. Each case with altered single-strand conformational polymorphism, analyzed by DNA sequencing and/or restriction analysis, showed the same alteration of codon 31, a C to A transversion encoding a change in amino acid sequence from serine to arginine (31Ser-->31Arg). DNA from the blood of 21 normal individuals showed the same alteration in WAF1/Cip1 in 4 cases (19%). Furthermore, paired normal tissue was available for 3 of 20 breast carcinomas with the Ser31Arg transversion. Normal DNA from all 3 cases showed the same 31Arg alteration as found in the tumor tissue. These results indicate that codon 31 is a polymorphic site and that the serine to arginine shift is a polymorphism. p21WAF1/Cip1 expression, identified by immunohistochemistry, was found to vary in a pattern that depended both on the tissue type and on the presence or absence of the codon 31 polymorphism. Using pair-wise comparisons in breast DCIS, we found higher protein expression in tumor nuclei as compared with benign stromal cell nuclei (P = 0.002) or normal ductal epithelium (P = 0.005). Invasive breast cancer specimens showed a trend in p21WAF1/Cip1 immunostaining similar to DCIS but did not reach statistical significance (P = 0.12). However, when cases with extensive desmoplastic reaction were excluded, a statistically significant association (P = 0.019) similar to that in DCIS was noted. In contrast to the breast tumors, ovarian carcinomas exhibited significantly greater p21WAF1/Cip1 expression in the benign stromal (fibroblast) nuclei surrounding the tumor than in the carcinoma cell nuclei (P = 0.016). Endometrial carcinoma revealed no difference in staining when comparing benign tissue with carcinoma (P = 0.99); however, unlike breast and ovarian carcinomas in which there was no correlation between p21WAF1/Cip1 expression and the presence or absence of the alteration at the 31st codon, endometrial carcinomas showed an increased percentage of immunopositive nuclei associated (P = 0.056) with 31Arg. These results demonstrate tissue-specific expression patterns of WAF1/Cip1 in different tumors which appears to be characteristic of the tumor type.

Breast Neoplasms↗
PubMed1996

Abrogation of transforming growth factor-beta type II receptor stimulates embryonic mouse lung branching morphogenesis in culture.

TGF-beta1 is a known inhibitor of branching morphogenesis when added exogenously to mouse embryonic lungs in culture. However, the issue of whether endogenous TGF-beta signaling has a function in the process of lung organogenesis is not completely resolved. We utilized immunoperturbation and antisense oligodeoxynucleotide inhibitory strategies to abrogate TGF-beta type II receptor function in embryonic mouse lungs undergoing branching morphogenesis in serumless explant culture. Antisera directed against a TGF-beta type II receptor N-terminal peptide that perturbs TGF-beta ligand-receptor binding increased branching by 70%. Similarly, antisense TGF-beta type II receptor oligodeoxynucleotides (40 microM) resulted in a 58% increase in branching, compared to scrambled and mismatched sequence controls, while TGF-beta, type II receptor mRNA and its protein expression levels were suppressed by 95 and 84%, respectively. Addition of exogenous TGF-beta1 did not overcome the stimulatory effects either of TGF-beta type II receptor immunoperturbation or of antisense oligodeoxynucleotide treatment on lung branching morphogenesis. Using in situ hybridization and immunohistochemistry, both TGF-beta type II receptor mRNA and protein were localized to the epithelium lining the developing airways, and to the surrounding mesenchyme, indicating that TGF-beta type II receptor is an important regulator of epithelial-mesenchymal interaction. Exogenous TGF-beta1 decreased cyclin A mRNA levels in control embryonic lung explants, while TGF-beta type II receptor antisense oligodeoxynucleotides prevented the downregulation of cyclin A mRNA expression by exogenous TGF-beta1. In addition, PCNA immunostaining of the primitive bronchial epithelium was increased in the presence of TGF-beta type II receptor antisense oligodeoxynucleotides either alone or together with exogenous TGF-beta1, whereas TGF-beta1 alone decreased PCNA staining. Thus, abrogation of TGF-beta type II receptor expression prevented TGF-beta1-induced epithelial cell G1 arrest. These results demonstrate, for the first time, that abrogation of the TGF-beta type II receptor stimulates embryonic lung organogenesis in culture and reverses the negative influence of endogenous TGF-beta signaling upon epithelial cell cycle progression.

Animals↗
PubMed1996

Transforming growth factor-beta type-II receptor signalling: intrinsic/associated casein kinase activity, receptor interactions and functional effects of blocking antibodies.

The transforming growth factor beta (TGF-beta) family of growth factors control proliferation, extracellular matrix synthesis and/ or differentiation in a wide variety of cells. However, the molecular mechanisms governing ligand binding, receptor oligomerization and signal transduction remain incompletely understood. In this study, we utilized a set of antibodies selective for the extracellular and intracellular domains of the TGF-beta type-II receptor as probes to investigate the intrinsic kinase activity of this receptor and its physical association in multimeric complexes with type-I and type-III receptors. The type-II receptor immuno-precipitated from human osteosarcoma cells exhibited autophosphorylation and casein kinase activity that was markedly stimulated by polylysine yet was insensitive to heparin. Affinity cross-linking of 125I-TGF-beta 1 ligand to cellular receptors followed by specific immunoprecipitation demonstrated that type-II receptors form stable complexes with both type-I and type-III receptors expressed on the surfaces of both human osteosarcoma cells and rabbit chondrocytes. Pretreatment of the cultured cells with an antibody directed against a distinct extracellular segment of the type-II receptor (anti-TGF-beta-IIR-NT) effectively blocked the 125I-TGF-beta labelling of type-I receptors without preventing the affinity labelling of type-II or type-III receptors, indicating a selective disruption of the type-I/type-II hetero-oligomers. The anti-TGF-beta-IIR-NT antibodies also blocked the TGF-beta-dependent induction of the plasminogen activator inhibitor (PAI-1) promoter observed in mink lung epithelial cells. However, the same anti-TGF-beta-IIR-NT antibodies did not prevent the characteristic inhibition of cellular proliferation by TGF-beta 1, as determined by [3H]thymidine incorporation into DNA. The selective perturbation of PAI-1 promoter induction versus cell-cycle-negative regulation suggests that strategic disruption of TGF-beta type-I and -II receptor interactions can effectively alter specific cellular responses to TGF-beta signalling.

Amino Acid Sequence↗
PubMed1996

Factor XII-induced mitogenesis is mediated via a distinct signal transduction pathway that activates a mitogen-activated protein kinase.

Clotting factor XII (Hageman factor) contains epidermal growth factor (EGF)-homologous domains and is reported to be a potent mitogen for human hepatoma (HepG2) cells. In this study, we tested whether factor XII exhibits growth factor activity on several other EGF-sensitive target cells, including fetal hepatocytes, endothelial cells, alveolar type II cells, and aortic smooth muscle cells. We found that factor XII significantly enhanced [3H]thymidine incorporation in aortic smooth muscle cells (SMCs) and all other cells tested. Tyrphostin, a growth factor receptor/tyrosine kinase antagonist, inhibited both EGF- and factor XII-induced responses. However, differences in the levels of magnitude of DNA synthesis, the observed synergism between EGF and factor XII, and the differential sensitivity to tyrphostin suggest that the EGF receptor and the factor XII receptor may be nonidentical. The factor XII-induced mitogenic response was achieved at concentrations that were 1/10th the physiologic range for the circulating factor and was reduced by popcorn inhibitor, a specific factor XII protease inhibitor. Treatment of aortic SMCs with factor XII, as well as activated factor XII, resulted in a rapid and transient activation of a mitogen-activated/extracellular signal-regulated protein kinase with peak activity/tyrosine phosphorylation observed at 5 to 10 min of exposure. Taken together, these data (i) confirm that clotting factor XII functions as a mitogenic growth factor and (ii) demonstrate that factor XII activates a signal transduction pathway, which includes a mitogen-activated protein kinase.

Animals↗
PubMed1996

Biochemical characterization of the human cyclin-dependent protein kinase activating kinase. Identification of p35 as a novel regulatory subunit.

The activation of cyclin-dependent protein kinases (Cdks) is dependent upon site-specific phosphorylation and dephosphorylation reactions, as well as positive and negative regulatory subunits. The human Cdk-activating protein kinase (Cak1) is itself a Cdc2-related cyclin-dependent protein kinase that associates with cyclin H. The present study utilized specific anti-Cak1 antibodies and immunoaffinity chromatography to identify additional Cak1-associated proteins and potential target substrates. Immunoprecipitation of metabolically labeled human osteosarcoma cells revealed a number of Cak1-associated proteins, including p95, p37 (cyclin H), and a 35-kDa protein that was further characterized herein. Microsequence analysis obtained after limited proteolysis revealed peptide fragments that are similar, but not identical to, human and yeast cyclins, thus identifying p35 as a cyclin-like regulatory subunit. The greatest sequence similarity of human p35 is with Mcs2, a yeast cyclin that is essential for cell cycle progression. Immunoaffinity chromatography performed under nondenaturing conditions afforded the isolation of enzymatically active Cak1 from cell lysates, enabling studies of kinase autophosphorylation and comparative substrate utilization. Immunoaffinity-purified Cak1 phosphorylated monomeric Cdc2 and Cdk2, but not Cdk4; the phosphorylation of both Cdc2 and Cdk2 were increased in the presence of recombinant cyclin A. These studies indicate that the Cak1 catalytic subunit, like Cdc2 and Cdk2, associates with multiple regulatory partners and suggests that subunit composition may be an important determinant of this multifunctional enzyme.

Amino Acid Sequence↗
PubMed1996

A cdc2-like kinase distinct from cdk5 is associated with neurofilaments.

An immunoprecipitation assay was used to identify protein kinases which are physically associated with neurofilaments (NF) in mouse brain extracts. Using this approach, we show that a cdc2-related kinase is associated with NF. The cdc2-related kinase was found to be distinct from cdk5 and the authentic cdc2 by a number of criteria. Firstly, it has a molecular mass on SDS-PAGE gels of 34 kDa, similar to that of cdc2, but differing from cdk5 (31 kDa). Secondly, it is not recognized by an antibody specific for cdk5. Thirdly, it is recognized by an antibody raised against the C-terminal region of authentic cdc2, but not by an antibody specific for the PSTAIRE motif. Using immunoblotting, we further show that the cdc2-related kinase copurifies with NF isolated from rat tissues. In vitro kinase assays further demonstrated that immunoprecipitated cdc2-related kinase phosphorylates recombinant NF-H protein. Phosphorylation of NF-H by the cdc2-like activity was not affected by 3 microM olomoucine but was inhibited by 10 microM of this kinase inhibitor. Phosphoamino acid analysis of in vitro phosphorylated NF-H indicates that the immunoprecipitated cdc2-related kinase phosphorylates serine residues.

Animals↗
PubMed1996

Engineering, expression and renaturation of targeted TGF-beta fusion proteins.

This study reports the expression, purification, and renaturation of biologically active Transforming Growth Factor-beta 1 (TGF-beta 1) fusion proteins from Escherichia coli (E. coli). A prokaryotic expression vector was engineered to produce tripartite fusion proteins consisting of (i) a purification tag, (ii) a protease-sensitive linker/collagen binding domain, and (iii) a cDNA sequence encoding the active fragment of human TGF-beta 1. The expressed fusion proteins TGF-B1-F1 and TGF-B1-F2, located in inclusion bodies, were solubilized with 8 M urea and renatured using a glutathione redox-coupled system and protracted dialysis under several experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion proteins on a Ni-NTA metal chelate column. The biological activity of the recombinant growth factor was demonstrated by its ability to inhibit mink lung (Mv1Lu) cell proliferation and/or to stimulate proliferation of NIH-3T3 mouse fibroblasts, where purified human platelet TGF-beta 1 served as a positive control. Purified TGF-B1-F1 and TGF-B1-F2 (collagen-binding) constructs exhibited anti-proliferative activities comparable to purified platelet TGF-beta 1, but at lower specific activities. Binding of the renatured TGF-B1-F2 fusion protein to collagen was demonstrated by stable binding on a collagen-conjugated Sephadex-G15 column. The high affinity binding was also demonstrated by the binding of 3H-collagen to the TGF-B1-F2 protein immobilized on a Ni-NTA column. The TGF-B1-F2 fusion protein bound to collagen coated surfaces with high affinity but exhibited comparatively lower biological activity than the fusion protein in solution, suggesting a potentially latent configuration. Taken together, these results demonstrate that biologically active TGF-beta 1 fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for its high-yield production, purification, and renaturation from microorganisms. Furthermore, these results support the concept that auxiliary domains may be used to modulate and/or target TGF-beta 1 for specific applications.

3T3 Cells↗
PubMed1995

Molecular cloning of CDK7-associated human MAT1, a cyclin-dependent kinase-activating kinase (CAK) assembly factor.

Mammalian CDK7 is a protein kinase identified as the catalytic subunit of cyclin-dependent kinase (CDK)-activating kinase and as an essential component of the transcription factor TFIIH that is involved in transcription initiation and DNA repair. We have identified in human cells a number of CDK7-associated cellular proteins that appear to fall into two classes based on their relative [35S] metabolic labeling intensity. One class of proteins present in CDK7 immunocomplexes as a minor fraction contains components of the TFIIH transcription complex such as p62 and p89ERCC3, whereas the other fraction contains four polypeptides (p35, p37Cyclin H, p75, and p95) that are stoichiometrically associated with CDK7. Whereas the levels of association of p35, p37Cyclin H, and p75 with CDK7 remain unchanged between density-arrested and proliferating Ewing sarcoma EW-1 cells, the association of p95 with CDK7 was significantly decreased as cells reached confluency. Through a large-scale immunopurification of CDK7 complexes and protein microsequencing, we have isolated a cDNA that encodes p35 and have shown that it is the human homologue of Mat1 that is involved in the assembly of CAK. MAT1 contains a highly conserved C3HC4 motif at its NH2 terminus, a characteristic feature shared among RING finger proteins. The human MAT1 gene expresses a single 1.6-kb transcript, the steady-state level of which, like CDK7 and cyclin H, varies significantly in different cell lines and in different terminally differentiated tissues.

Amino Acid Sequence↗
PubMed1995

Retroviral vector-mediated gene transfer of antisense cyclin G1 (CYCG1) inhibits proliferation of human osteogenic sarcoma cells.

Genetic changes found in human osteogenic sarcoma cells, including loss of the p53 and Rb tumor suppressor elements and overexpression of the cyclin G1 (CYCG1) proto-oncogene, suggest the potential of gene transfer as a treatment for metastatic disease. In this study, we examined the effects of antisense cyclin G1, in comparison with antisense cyclin D1 (CYCD1) and enforced expression of the universal cyclin-dependent kinase inhibitor p21WAF1/CIP1 on the proliferation of human MG-63 osteosarcoma cells. Retroviral vectors bearing antisense CYCG1 as well as antisense CYCD1 and WAF1/CIP1 (in sense orientation) driven by the Moloney murine leukemia virus long terminal repeat promoter inhibited the growth and/or survival of transduced MG-63 cells in 2-7 day cultures. This represents the first demonstration that cyclin G1 is essential for the survival and/or growth of human osteosarcoma cells. Cytostatic and cytopathic effects were accompanied by a significant increase in the incidence of apoptosis, as determined by immunocytochemical analysis of DNA fragmentation. Furthermore, transduction of MG-63 cells with a retroviral vector bearing the suicide gene, herpes simplex thymidine kinase (HStk), induced cell death on treatment with ganciclovir, exhibiting pronounced bystander effects. Taken together, the data affirm the feasibility of modulating inducible cell cycle control enzymes as a potential gene therapy approach in the clinical management of osteogenic sarcoma.

Apoptosis↗
PubMed1995

Detection of a Cdc2-related kinase associated with Alzheimer paired helical filaments.

By immunocytochemical staining and Western blotting, we detected a Cdc2-related kinase in human brains. The kinase is recognized by antibodies against the carboxyl and the amino termini of p34Cdc2 but is not recognized by antibodies against the PSTAIRE motif. It is slightly smaller than p34Cdc2 in molecular mass (approximately 33 kd). This 33-kd Cdc2-related kinase is present in intracellular neurofibrillary tangles in neurons of elderly humans and in Alzheimer's disease, and it is associated with paired helical filaments (PHF) from Alzheimer's disease brains. Unlike the antibodies to the carboxyl and amino termini of p34Cdc2, antibodies to an abundant brain Cdc2-related kinase PSSLARE/Cdk5 did not immunolabel Alzheimer's disease neurofibrillary lesions. PHF preparations were demonstrated to contain kinases capable of phosphorylating histone H1, PHF-Tau, and a synthetic peptide (VAVVRTPPKSPSSAK). By virtue of its physical association with PHF, the 33-kd Cdc2-related kinase may play a role in transforming normal Tau proteins to PHF-Tau characteristic of Alzheimer's disease.

Alzheimer Disease↗
PubMed1994

Phosphorylation and inactivation of protein phosphatase 1 by cyclin-dependent kinases.

Protein phosphatase 1 and protein phosphatase 2A contain potential phosphorylation sites for cyclin-dependent kinases. In the present study we found that rabbit skeletal muscle protein phosphatase 1, as well as recombinant protein phosphatase 1 alpha and protein phosphatase 1 gamma 1, but not protein phosphatase 2A, was phosphorylated and inhibited by cdc2/cyclin A and cdc2/cyclin B. Phosphopeptide mapping and phospho amino acid analysis suggested that the phosphorylation site was located at a C-terminal threonine. Neither cdc2/cyclin A nor cdc2/cyclin B phosphorylated an active form of protein phosphatase 1 alpha in which Thr-320 had been mutated to alanine, indicating that the phosphorylation occurred at this threonine residue. Furthermore, protein phosphatase 1, but not protein phosphatase 2A, activity was found to change during the cell cycle of human MG-63 osteosarcoma cells. The observed oscillations in protein phosphatase 1 activity during the cell cycle may be due, at least in part, to phosphorylation of protein phosphatase 1 by cyclin-dependent kinases. Together, the results suggest a mechanism for direct regulation of protein phosphatase 1 activity.

Amino Acids↗