Biomedical subjects

L T Chow

Publications and source records attributed to L T Chow.

At least 37 records · Page 2Linked to original sources

PubMed2000

Casein kinase II phosphorylation of the human papillomavirus-18 E7 protein is critical for promoting S-phase entry.

The human papillomavirus type 18 E7 protein subverts the pRb/E2F pathway to promote S-phase reentry by postmitotic, differentiated primary human keratinocytes in support of viral DNA amplification. We prepared a panel of HPV-18 E7 mutations in pRb binding or in casein kinase II (CKII) phosphorylation. Our results showed that the ability of E7 binding to pRb correlated with the activation of DNA polymerase alpha or cyclin E to various extents in differentiated keratinocytes of organotypic cultures but was insufficient to induce the proliferating cell nuclear antigen. Proteins mutated in the CKII recognition sequence or in one or both serine substrates (S32 and S34) bound pRb in vitro, but only those with negative charges at these two residues induced proliferating cell nuclear antigen effectively. Nevertheless, unscheduled cellular DNA synthesis occurred very inefficiently relative to the wild-type E7, if at all. Thus, both pRb binding and CKII phosphorylation of E7 are critical for activating cellular genes essential for S-phase entry.

Amino Acid Motifs↗

PubMed1999

Conditional expression of the ErbB2 oncogene elicits reversible hyperplasia in stratified epithelia and up-regulation of TGFalpha expression in transgenic mice.

The ErbB2 receptor tyrosine kinase (RTK) is expressed in basal cells of squamous epithelia and the outer root sheath of hair follicles. We previously showed that constitutive expression of activated ErbB2 directed to these sites in the skin by the keratin 14 (K14) promoter produces prominent hair follicle abnormalities and striking skin hyperplasia in transgenic mice. However, perinatal lethality precluded the establishment of a transgenic line for analysis of ErbB2 function in adult animals. To investigate the significance of ErbB2 signaling in epithelial tissues during and post development, we developed a K14-rtTA/TetRE-ErbB2 'Tet-On' bitransgenic mouse system. These mice were normal until the ErbB2 transgene was induced by exposure to doxycycline (Dox). Prenatal induction resulted in perinatal death. Postnatally, ErbB2 transgene expression was observed at 4 h after the initiation of Dox, and reached a plateau at 24 h. Skin hyperplasia followed after 2 days and these changes reverted to normal upon Dox withdrawal. In adults, as in the neonates, prolonged ErbB2 induction caused prominent skin and hair follicle hyperplasias. Severe hyperplasias in the cornea, eye lids, tongue and esophagus were also observed. ErbB2 transgene induction was accompanied by increased expression of TGFalpha, a ligand of epidermal growth factor receptor (EGFR), and to a lesser extent, EGFR, further enhancing RTK signal transduction. We conclude that ErbB2 plays important roles in both development and maintenance of hair follicles and diverse squamous epithelia and that this ligand-inducible and tissue-specific 'Tet-On' transgenic mouse system provides a means to study transgenes with perinatal toxicity.

Animals↗

PubMed1999

Human papillomavirus DNA replication. Interactions between the viral E1 protein and two subunits of human dna polymerase alpha/primase.

Papovaviruses are valuable models for the study of DNA replication in higher eukaryotic organisms, as they depend on host factors for replication of their DNA. In this study we investigate the interactions between the human papillomavirus type 11 (HPV-11) origin recognition and initiator protein E1 and human polymerase alpha/primase (pol alpha/primase) subunits. By using a variety of physical assays, we show that both 180- (p180) and 70-kDa (p70) subunits of pol alpha/primase interact with HPV-11 E1. The interactions of E1 with p180 and p70 are functionally different in cell-free replication of an HPV-11 origin-containing plasmid. Exogenously added p180 inhibits both E2-dependent and E2-independent cell-free replication of HPV-11, whereas p70 inhibits E2-dependent but stimulates E2-independent replication. Our experiments indicate that p70 does not physically interact with E2 and suggest that it may compete with E2 for binding to E1. A model of how E2 and p70 sequentially interact with E1 during initiation of viral DNA replication is proposed.

Animals↗

PubMed1999

A distal element in the HPV-11 upstream regulatory region contributes to promoter repression in basal keratinocytes in squamous epithelium.

In benign squamous lesions and in organotypic epithelial cultures, the human papillomavirus (HPV) E6 and E7 genes are transcriptionally up-regulated in differentiated, spinous keratinocytes. We previously identified sequence elements in the enhancer-promoter regions of HPV types 18 and 11 important for this promoter regulation by using the bacterial LacZ reporter gene in stratified raft cultures of primary human keratinocytes (PHKs) or in submerged, proliferating cultures acutely transduced with recombinant retroviruses. Notably, mutations in the promoter-proximal Sp1, Oct1, and AP1 sites each significantly reduce reporter activity in differentiated cells, indicating that the bound factors are transcription transactivators. In the present study, we performed further mutagenesis on distal motifs in the HPV-11 regulatory region in PHKs in submerged and raft cultures. Mutations in an AP2-like site, three individual NF-1 sites, or five NF-1 sites collectively reduced promoter activity slightly in differentiated cells. A mutation in a putative glucocorticoid response element had no discernable effect in the presence or the absence of dexamethasone. However, mutations in a C/EBP binding site, especially the distal site, strikingly up-regulated reporter gene expression, particularly in basal and lower spinous cells, implicating bound protein as a transcription repressor. Collectively, these results demonstrate that the overall differentiation-dependent papillomaviral gene expression observed in vivo and in vitro involves promoter repression in the lower strata and activation in the upper, differentiated strata.

Binding Sites↗

PubMed1999

Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication.

We have identified the human papillomavirus (HPV) DNA replication initiation protein E1 as a tight-binding substrate of cyclin E/cyclin-dependent kinase (Cdk) complexes by using expression cloning. E1, a DNA helicase, collaborates with the HPV E2 protein in ori-dependent replication. E1 formed complexes with cyclin E in insect and mammalian cells, independent of Cdks and E2. Additional cyclins, including A-, B-, and F-type (but not D-type), interacted with the E1/E2 complex, and A- and E-type cyclin kinases were capable of phosphorylating E1 and E2 in vitro. Association with cyclins and efficient phosphorylation of E1 required the presence of a cyclin interaction motif (the RXL motif). E1 lacking the RXL motif displayed defects in E2-dependent HPV ori replication in vivo. Consistent with a role for Cdk-mediated phosphorylation in E1 function, an E1 protein lacking all four candidate Cdk phosphorylation sites still associated with E2 and cyclin E but was impaired in HPV replication in vitro and in vivo. Our data reveal a link between cyclin/Cdk function and activation of HPV DNA replication through targeting of Cdk complexes to the E1 replication-initiation protein and suggest a functional role for E1 phosphorylation by Cdks. The use of cyclin-binding RXL motifs is now emerging as a major mechanism by which cyclins are targeted to key substrates.

Cloning, Molecular↗

PubMed1999

Sonographic measurement of splenic length: correlation with measurement at autopsy.

PURPOSE: The purpose of this study was to determine the correlations between the sonographic measurement of splenic length and the actual splenic length, volume, and weight. METHODS: Before autopsy, sonographic measurements of splenic length were obtained in 30 cadavers, and these values were compared with the actual length, volume, and weight of the spleen at autopsy. RESULTS: There were clear linear correlations between maximum sonographic length and actual length (r = 0.831), volume (r = 0.817), and weight (r = 0.810). CONCLUSION: This study shows that a single, simple sonographic measurement gives a clinically useful indication of true splenic size.

Aged↗

PubMed1999

Favorable scanning electron microscopic findings of stapled saphenous vein-carotid artery anastomoses.

BACKGROUND: In the progress toward "off-pump" endoscopic coronary artery surgical procedures, new techniques for coronary artery anastomoses are being developed. One such approach is the use of nonpenetrating titanium clips. We evaluated the quality of anastomoses achieved using this technique in a porcine model of saphenous vein-carotid artery grafting using scanning electron microscopy. METHODS: Bilateral saphenous vein-carotid artery interposition grafts were implanted in 10 "white race" pigs, using the nonpenetrating clips in one side of the neck and conventional hand suturing on the opposite side. One week after operation, the grafts were harvested. RESULTS: All grafts were patent 7 days after operation, and 40 anastomoses underwent scanning electron microscopic study. In all samples, the luminal surfaces of both the carotid artery and vein graft were covered by a continuous layer of endothelial cells up to the anastomosis. Anastomotic sites in all clipped samples and most of the sutured anastomoses were completely endothelialized, and anastomotic clefts were indistinguishable. However, in 25% of sutured specimens, the suture material remained clearly visible inside the lumen of the vessel, and the subendothelial matrix remained exposed, with extensive fibrin, red blood cells, and platelet deposition on its surface. CONCLUSIONS: Because the endothelial coverage consistently appeared to be complete and the subendothelial matrix was not exposed, it is likely that the risk of early anastomotic thrombosis is reduced by using the nonpenetrating titanium clips.

Anastomosis, Surgical↗

PubMed1999

Left and right ventricular collagen type I/III ratios and remodeling post-myocardial infarction.

BACKGROUND: Types I and III collagen have different physical properties, and an increase of type I/III ratio can have a deleterious impact on myocardial compliance and left and right ventricular diastolic function. Post-myocardial infarction, these changes in collagen types may be relevant to the remodeling process and the development of heart failure. METHODS AND RESULTS: In the rat coronary ligation heart failure model, we studied the time course of changes in types I and III and total collagen levels over 10 weeks postinfarction. Collagen types were separately quantified in the left (LV) and right ventricles (RV) by computerized morphometry and standard immunohistochemistry techniques, and also by hydroxyproline analysis, and these were correlated with hemodynamic changes. Compared with sham-operated rats, total collagen level increased 2.5- to 2.9-fold and 1.7- to 2.9-fold in the noninfarcted areas (NIAs) of the LV and RV, respectively, over the 10-week period and showed a good relation with changes in hydroxyproline content (r2 = 0.62; P < .0001). In the NIAs of both the LV and RV, type III collagen level showed a transient twofold increase at 2 weeks, which declined to normal at 4 weeks. Type I collagen level increased twofold at 4 weeks in the NIA of the LV and remained elevated at 10 weeks. In the RV, type I collagen level increased 2.7-fold to a peak at 4 weeks and declined gradually to 1.7 times baseline at 10 weeks. The patterns of change in type I collagen level in the RV correlated with the changes in LV end-diastolic pressure (r = 0.73; P < .0001) and RV weight to body weight ratio (r = 0.73; P < .0001). CONCLUSION: There is a relative greater increase of type I collagen level in the NIA and RV postinfarction, and this may lead to left and right ventricular dysfunction. Separate mechanisms might be involved in the induction of the different types of collagen deposition, with type I collagen levels apparently closely correlating with hemodynamic stress.

Analysis of Variance↗

PubMed1999

Chronic recurrent multifocal osteomyelitis: a great clinical and radiologic mimic in need of recognition by the pathologist.

The spectrum of histopathologic changes in four cases of chronic recurrent multifocal osteomyelitis encountered in our orthopedic outpatient clinic in the past 3 years was studied in conjunction with clinical and radiologic findings. All presented with pain with or without swelling in the affected region. Radiographically, the appearance of the lesions varied from a mixed picture of bone lysis and sclerosis with expansion to sclerosis alone to bone collapse. Bone scintigraphy demonstrated asymptomatic and separate foci of activity in all cases. Prior to biopsy, the clinical and radiologic differential diagnoses included Ewing's sarcoma, metastatic neuroblastoma, hematolymphoid malignancy, Langerhans cell histiocytosis and chronic infection, notably tuberculosis. The spectrum of histopathologic changes ranged from acute (acute inflammatory infiltration, active bone resorption and necrosis, reactive bone formation) to subacute (predominantly lymphocytic and plasma cell infiltration) to chronic inflammation (fibroblastic organization and bony sclerosis). Histologic changes correlated poorly with clinical features, but relatively well with radiologic findings. Lesional excision was performed in one case, cortical saucerization in another, while the final two cases received supportive treatment. All remained well 18-21 months post-therapy. Chronic recurrent multifocal osteomyelitis is a great clinical and radiologic mimic, which merits recognition by the pathologist. Awareness of the spectrum of histologic features encountered enables a correct diagnosis to be made in the appropriate clinical setting. The patient can thus be reassured of a favorable prognosis.

Adolescent↗

PubMed1999

Human papillomavirus DNA replication compartments in a transient DNA replication system.

Many DNA viruses replicate their genomes at nuclear foci in infected cells. Using indirect immunofluorescence in combination with fluorescence in situ hybridization, we colocalized the human papillomavirus (HPV) replicating proteins E1 and E2 and the replicating origin-containing plasmid to nuclear foci in transiently transfected cells. The host replication protein A (RP-A) was also colocalized to these foci. These nuclear structures were identified as active sites of viral DNA synthesis by bromodeoxyuridine (BrdU) pulse-labeling. Unexpectedly, the great majority of RP-A and BrdU incorporation was found in these HPV replication domains. Furthermore, E1, E2, and RP-A were also colocalized to nuclear foci in the absence of an origin-containing plasmid. These observations suggest a spatial reorganization of the host DNA replication machinery upon HPV DNA replication or E1 and E2 expression. Alternatively, viral DNA replication might be targeted to host nuclear domains that are active during the late S phase, when such domains are limited in number. In a fraction of cells expressing E1 and E2, the promyelocytic leukemia protein, a component of nuclear domain 10 (ND10), was either partially or completely colocalized with E1 and E2. Since ND10 structures were recently hypothesized to be sites of bovine papillomavirus virion assembly, our observation suggests that HPV DNA amplification might be partially coupled to virion assembly.

Cell Nucleus↗

PubMed1999

Trichostatin A up-regulates human papillomavirus type 11 upstream regulatory region-E6 promoter activity in undifferentiated primary human keratinocytes.

Human papillomavirus (HPV) gene expression in squamous epithelia is differentiation dependent in benign patient lesions and in organotypic raft cultures of primary human keratinocytes (PHKs). Using the lacZ reporter in raft cultures, we previously showed that this transcriptional regulation of the HPV type 11 (HPV-11) enhancer-promoter located in the upstream regulatory region (URR) appears to have resulted from coordination between the transcription transactivators AP1, Oct1, and Sp1 in differentiated upper strata and the repressor C/EBP in proliferating basal cells. We report here that trichostatin A, a specific inhibitor of histone deacetylase, dramatically stimulated reporter gene activity from the wild-type HPV-11 URR or the C/EBP mutation in PHKs grown in undifferentiated submerged cultures. In epithelial raft cultures, up-regulation occurred predominantly in basal and parabasal strata; this effect was promoter specific, as expression of the lacZ reporter gene driven by the murine leukemia virus long terminal repeat (LTR), the keratin 14 promoter, or the involucrin promoter was not altered, nor was expression of endogenous keratin 10 and profilaggrin affected. However, the responses were not cell type or species specific, as identical results were observed for both HPV-11 URR-lacZ and LTR-lacZ in murine retrovirus producer cell lines of fibroblast origin.

CCAAT-Enhancer-Binding Proteins↗

PubMed1999

Components of human papillomavirus that activate transcription and support plasmid replication in human airway cells.

Human papillomaviruses (HPVs) such as types 6 and 11 can establish lifelong infections in airway epithelial cells in patients, and long-term infection can lead to pulmonary involvement and death. The mechanisms underlying this persistence depend on both the transcriptional activity of the viral enhancers and promoters and the ability of this virus to maintain its double-stranded circular DNA genome in infected tissues. We investigated the transcription and replication properties of HPV sequence elements and protein products in a human airway cell line. We showed that incorporation of the upstream regulatory region and cotransfection with expression vectors of two virus-encoded proteins, E1 and E2, conferred approximately 5,000-fold stimulation of reporter gene expression. Transient plasmid replication in transfected human airway cells and lungs of FVB/N-C57BL/6 mice was demonstrated by a modified transient replication assay. These results have important implications for viral pathogenesis in airway cells and the potential of HPV-based replicons for gene transfer into airway epithelium.

Animals↗

PubMed1999

Assessing chemotherapy response of squamous cell oesophageal carcinoma with spiral CT.

45 patients with squamous cell carcinoma of the oesophagus were examined prior to, and following, pre-operative chemotherapy by spiral CT. Oesophageal CT was performed following gaseous distention of the oesophagus. TNM stage and perceived resectability on CT before and after chemotherapy were compared and related to surgical resectability and pathological staging. T-stage changed in 26% and N-stage changed in 9% of tumours after chemotherapy. Post-chemotherapy CT predicted pathological T-stage with an accuracy of 88% and N-stage with an accuracy of 84%. Six of 14 tumours considered irresectable on CT pre-chemotherapy were considered resectable on post-chemotherapy CT. Five of these six tumours were resectable at surgery. Post-chemotherapy CT predicted surgical resectability with an accuracy of 88%, the main pitfall being underestimation and overestimation of tracheobronchial invasion. CT prediction of chemotherapy response as judged by change in tumour volume was compared with a quantitative pathological assessment of chemotherapy response. 93% of oesophageal tumours changed volume after chemotherapy with 51% having a volume reduction of > or = 50%. However, no correlation was found between tumour volume reduction on serial CT examinations and either a quantitative pathological assessment of tumour response or patient survival.

Carcinoma, Squamous Cell↗

PubMed1999

Concordant induction of cyclin E and p21cip1 in differentiated keratinocytes by the human papillomavirus E7 protein inhibits cellular and viral DNA synthesis.

Productive infections by human papillomaviruses (HPVs) occur only in differentiated keratinocytes in squamous epithelia in which the HPV E7 protein reactivates the host DNA replication machinery to support viral DNA replication. In a fraction of the differentiated keratinocytes, E7 also posttranscriptionally induces p21Cip1, which is distributed in a mutually exclusive manner with unscheduled cellular DNA synthesis. In this study, double immunofluorescence labeling unexpectedly revealed that E7 caused a concordant accumulation of both cyclin E and p21Cip1 to high levels in patient papillomas and in organotypic cultures of primary human keratinocytes. The induction of cyclin E is mutually exclusive with unscheduled cellular DNA synthesis or abundant viral DNA. These novel virus-host interactions in differentiated keratinocytes are in contrast to previous observations made in submerged proliferating cultures, in which HPV E7 induces cyclin E and overcomes p21Cip1 inhibition of S-phase entry. We propose that an appropriately timed induction of cyclin E/cyclin-dependent kinase 2 by HPV E7 in postmitotic cells enables S-phase reentry and HPV DNA amplification, whereas prematurely induced cyclin E stabilizes p21Cip1 protein, which then inhibits cyclin E/cyclin-dependent kinase 2. Consequently, cyclin E and p21Cip1 both fail to turn over, and DNA synthesis does not occur.

Basal Ganglia↗

PubMed1999

Oncogene expression cloning by retroviral transduction of adenovirus E1A-immortalized rat kidney RK3E cells: transformation of a host with epithelial features by c-MYC and the zinc finger protein GKLF.

The function of several known oncogenes is restricted to specific host cells in vitro, suggesting that new genes may be identified by using alternate hosts. RK3E cells exhibit characteristics of epithelia and are susceptible to transformation by the G protein RAS and the zinc finger protein GLI. Expression cloning identified the major transforming activities in squamous cell carcinoma cell lines as c-MYC and the zinc finger protein gut-enriched Kruppel-like factor (GKLF)/epithelial zinc finger. In oral squamous epithelium, GKLF expression was detected in the upper, differentiating cell layers. In dysplastic epithelium, expression was prominently increased and was detected diffusely throughout the entire epithelium, indicating that GKLF is misexpressed in the basal compartment early during tumor progression. The results demonstrate transformation of epithelioid cells to be a sensitive and specific assay for oncogenes activated during tumorigenesis in vivo, and identify GKLF as an oncogene that may function as a regulator of proliferation or differentiation in epithelia.

Adenovirus E1A Proteins↗

PubMed1998

Human Hsp70 and Hsp40 chaperone proteins facilitate human papillomavirus-11 E1 protein binding to the origin and stimulate cell-free DNA replication.

Human papillomavirus replication initiator, the E1 helicase, binds weakly to the origin of DNA replication. Purified human chaperone proteins Hsp70 and Hsp40 (HDJ-1 and HDJ-2) independently and additively enhanced E1 binding to the origin. The interaction between E1 and Hsp70 was transient and required ATP hydrolysis, whereas Hsp40 bound to E1 directly and remained in the complex. A peptide of 20 residues spanning the HPD loop and helix II of the J domain of YDJ-1 also stimulated E1 binding to the origin, alone or in combination with Hsp70 or Hsp40. A mutated peptide (H34Q) had a reduced activity, while an adjacent or an overlapping peptide had no effect. Neither Hsp70 nor the J peptide altered the E1/DNA ratio in the complex. Electron microscopy showed that E1 mainly bound to DNA as a hexamer. In the presence of Hsp40, E1 primarily bound to DNA as a dihexamer. Preincubation of chaperones with viral E1 and template shortened the lag time and increased replication in a cell-free system. Since two helicases are essential for bidirectional replication of human papillomavirus DNA, these results demonstrate that, as in prokaryotes, chaperones play an important role in the assembly of preinitiation complexes on the origin.

Carrier Proteins↗

PubMed1998

Post-transcriptional induction of p21cip1 protein by human papillomavirus E7 inhibits unscheduled DNA synthesis reactivated in differentiated keratinocytes.

Productive infection by human papillomaviruses (HPVs) occurs only in differentiated squamous epithelial cells in papillomas, condylomata, and low grade intraepithelial neoplasias. Host DNA replication is reactivated in a fraction of terminally differentiated keratinocytes in benign human lesions and in organotypic raft cultures of primary human keratinocytes (PHKs) transduced with retroviruses expressing HPV-18 E7 oncogene from its native upstream regulatory region (URR). Thus the natural function of E7 protein, which inactivates pRB family proteins, is to induce host genes essential to support viral DNA replication in post-mitotic cells. Using this raft culture model system, we show that HPV-18 URR-E7 induces the universal cyclin-dependent kinase inhibitor p21cip1 protein in a fraction of differentiated PHKs. Induction is mediated by posttranscriptional mechanisms independent of p53. Double immunofluorescence studies demonstrate that, in raft cultures and in laryngeal papillomas, p21cip1 induction and reactivated host DNA synthesis take place in a mutually exclusive manner in PCNA-positive, differentiated keratinocytes. We suggest that p21cip1 induction effectively blocks unscheduled DNA synthesis reactivated by E7. These results begin to explain the inverse relationship between p21cip1 induction and HPV activities previously observed in a spectrum of benign lesions regardless of HPV types present.

Antigens, Neoplasm↗

PubMed1998

Intracortical osteosarcoma.

Intracortical osteosarcomas originate in the bone cortex and represent the rarest type of osteosarcoma. We describe the clinical, radiological and histological features of an intracortical osteosarcoma occurring in the femur of a young man and discuss the pertinent features of this tumour compared to those previously reported.

Adult↗