Longitudinal sampling is required to maximize detection of intrahost A/H3N2 virus variants.

Seasonal human influenza viruses continually change antigenically to escape from neutralizing antibodies. It remains unclear how genetic variation in the intrahost virus population and selection at the level of individual hosts translates to the fast-paced evolution observed at the global level because emerging intrahost antigenic variants are rarely detected. We tracked intrahost variants in the hemagglutinin and neuraminidase surface proteins using longitudinally collected samples from 52 patients infected by A/H3N2 influenza virus, mostly young children, who received oseltamivir treatment. We identified emerging putative antigenic variants and oseltamivir-resistant variants, most of which remained detectable in samples collected at subsequent days, and identified variants that emerged intrahost immediately prior to increases in global rates. In contrast to most putative antigenic variants, oseltamivir-resistant variants rapidly increased to high frequencies in the virus population. Importantly, the majority of putative antigenic variants and oseltamivir-resistant variants were first detectable four or more days after onset of symptoms or start of treatment, respectively. Our observations demonstrate that de novo variants emerge, and may be positively selected, during the course of infection. Additionally, based on the 4-7 days post-treatment delay in emergence of oseltamivir-resistant variants in six out of the eight individuals with such variants, we find that limiting sample collection for routine surveillance and diagnostic testing to early timepoints after onset of symptoms can potentially preclude detection of emerging, positively selected variants.

Influences of alignment and obesity on knee joint loading in osteoarthritic gait.

OBJECTIVE: To determine the influences of frontal plane knee alignment and obesity on knee joint loads in older, overweight and obese adults with knee osteoarthritis (OA). METHODS: Cross-sectional investigation of alignment and obesity on knee joint loads using community dwelling older adults (age ≥ 55 years; 27 kg m(-2) ≥ body mass or body mass index (BMI) ≤ 41 kg m(-2); 69% female) with radiographic knee OA that were a subset of participants (157 out of 454) enrolled in the Intensive Diet and Exercise for Arthritis (IDEA) clinical trial. RESULTS: A higher BMI was associated with greater (P = 0.0006) peak knee compressive forces [overweight, 2411 N (2182, 2639), class 1 obesity, 2772 N (2602, 2943), class 2+ obesity, 2993 N (2796, 3190)] and greater (P = 0.004) shear forces [overweight, 369 N (322, 415), class 1 obesity, 418 N (384, 453), class 2+ obesity, 472 N (432, 513)], independent of alignment, and varus alignment was associated (P < 0.0001) with greater peak external knee adduction moments, independent of BMI [valgus, 18.7 Nm (15.1, 22.4), neutral, 27.7 Nm (24.0, 31.4), varus, 37.0 Nm (34.4, 39.7)]. CONCLUSION: BMI and alignment were associated with different joint loading measures; alignment was more closely associated with the asymmetry or imbalance of loads across the medial and lateral knee compartments as reflected by the frontal plane external adduction moment, while BMI was associated with the magnitude of total tibiofemoral force. These data may be useful in selecting treatment options for knee OA patients (e.g., diet to reduce compressive loads or bracing to change alignment).

Biomechanical responses of the back of riding horses to water treadmill exercise.

There is a lack of evidence for the presumed beneficial effects of water treadmills on the movement of the horse's back. The aim of the study was to evaluate the effects of water treadmill exercise on axial rotation (AR), lateral bending (LB) and pelvic flexion (PF) in horses. The back kinematics of a group of riding horses were studied at the walk in a water treadmill at different depths of water (hoof, fetlock, carpus, elbow and shoulder joint levels) over a period of 10 days. Skin markers were placed at anatomical locations on the back. AR, LB and PF were measured on days 1 and 10 using two high-speed video cameras. There was a significant increase in AR compared to baseline at the level of the carpus and at higher water levels, whereas LB was significantly lower than baseline values at water levels that reached the elbow and shoulder joints. PF was significantly higher than baseline values at each water depth other than hoof water depth. At increasing water depths, there were significant increases in flexion and rotation of the back. At the highest water levels, there was reduced bending of the back. After 10 days, horses exhibited more bending of the back.

The seroprevalence of Lawsonia intracellularis in horses in The Netherlands.

Equine proliferative enteropathy caused by Lawsonia intracellularis is an emerging disease of weanling foals and affects their growth and development. The prevalence of Lawsonia intracellularis in The Netherlands is not known. The aim of the study was to investigate the seroprevalence of Lawsonia intracellularis in horses in The Netherlands. Blood samples were taken from healthy foals before and after weaning and from healthy yearlings and mature horses on farms throughout The Netherlands. These samples were analysed for the presence of Lawsonia intracellularis-specific antibodies with a blocking ELISA. White blood cell count, packed cell volume, and total protein concentration were also measured in all foals. Information regarding housing, pasture access, and contact with pig manure on the premises was obtained for all animals. The prevalence of Lawsonia intracellularis antibodies in foals increased significantly from 15% before weaning to 23% after weaning (p = 0.019); it was 89% in yearlings and 99% in horses older than 2 years. There was no significant difference in seroprevalence between the pasture-kept and stable-confined adult horses (97% and 100%, respectively), and there was no significant influence of contact with pig manure. None of the sampled animals showed clinical disease. In conclusion, the results suggest that Lawsonia intracellularis is widespread in The Netherlands and that seropositivity is not necessarily associated with clinical problems. The high seroprevalence in adult horses suggests long-term persistence of antibodies against Lawsonia intracellularis or constant exposure to the bacterium.

BAG-1, a novel Bcl-2-interacting protein, activates expression of human JC virus.

Transcription of the human polyomavirus JC virus (JCV) genome is regulated by cellular proteins and the large tumour (T) antigen. Earlier studies led to the identification of nuclear factor-1 (NF-1)-binding sites in the JCV enhancer by DNase I protection assays of extracts from retinoic acid (RA)-differentiated P19 embryonal carcinoma (EC) cells. In this study, a cDNA clone that encodes a protein capable of binding to the JCV NF-1 sites was isolated from an RA-differentiated EC cell cDNA library. Sequence analysis revealed that the cDNA isolated was identical to the previously described Bcl-2-interacting protein BAG-1 (Bcl-2-associated athano gene-1). Results from RNA studies indicated that BAG-1 is expressed in several cell types. Co-transfection of a recombinant BAG-1 expression plasmid with JCV promoters indicated that BAG-1 stimulates transcription of the JCV(E) promoter and to a lesser extent the JCV(L) promoter. Mutations in the NF-1 sites in the JCV(E) promoter eliminated the activation by BAG-1. Thus, BAG-1 is a novel transcription factor that may play a role in JCV expression.

Human BAG-1/RAP46 protein is generated as four isoforms by alternative translation initiation and overexpressed in cancer cells.

Previously, a Bcl-2-interacting protein, BAG-1, was cloned from mouse cells and was shown to interact with several other proteins and to be important for inhibition of apoptosis. Human BAG-1 (hBAG-1) cDNA, recently isolated by us and two other groups, has been shown to be identical to a hormone receptor-binding protein, RAP46. However, different molecular masses of hBAG-1 protein products were noted by these three groups. Here we demonstrated that hBAG-1 protein was expressed as four isoforms, designated p50, p46, p33 and p29, with apparent molecular masses of 50 kDa, 46 kDa, 33 kDa and 29 kDa, respectively. Deletion, site-directed mutagenesis and in vitro transcription/translation analysis showed that the four protein products of hBAG-1 were expressed by alternative initiation from four different start codons through a leaky scanning mechanism. Furthermore, we demonstrated that the distinct forms of hBAG-1 have different subcellular localizations, suggesting that they may have distinct functions in the cells. Characterization of hBAG-1 RNA and protein also showed that hBAG-1 was overexpressed in human cervical, breast and lung cancer cell lines. Taken together, these data clarify the conflicting observations reported in the literature and suggest that hBAG-1 is expressed as four forms of protein products, which may play a differential role in apoptosis and oncogenesis of human cells.

Enhanced expression of anti-apoptotic proteins in human papillomavirus-immortalized and cigarette smoke condensate-transformed human endocervical cells: correlation with resistance to apoptosis induced by DNA damage.

Apoptosis plays an important role in various biological processes including embryogenesis, differentiation, homeostasis, and oncogenesis. We have developed a system composed of primary human endocervical cells (HEN), HEN immortalized by human papillomavirus (HPV) type 16, and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). To understand the role of apoptosis in the multistep oncogenesis of human cervical cells, we examined the expression of apoptosis-associated proteins in our in vitro model system. The results showed no significant difference in the levels of apoptosis-inducing proteins bak and bax among all the cell types examined. On the other hand, the levels of apoptosis-inhibiting proteins bcl-2, bcl-xL and BAG-1 increased progressively after immortalization and transformation. The p53 protein level decreased in the HPV16-immortalized HEN and increased in one of two lines of the CSC-transformed HEN. Further, the increased levels of apoptosis-inhibiting proteins in the HPV16-immortalized and the CSC-transformed HEN correlated with progressively increased resistance of these cells to apoptosis induced by staurosporine or cisplatin. This study provided the first evidence that overexpression of apoptosis-inhibiting proteins is important for both multistep oncogenesis and resistance of human endocervical cells to apoptosis induced by DNA-damaging reagents.

Identification and characterization of a JC virus pentanucleotide repeat element binding protein: cellular nucleic acid binding protein.

The JC virus (JCV) control region contains AGGGAAGGGA, the tandem pentanucleotide repeat element (Pnt2). Several proteins specifically interacted via Pnt2 to regulate the expression of JCV early promoter-enhancer (JCV(E)) or late promoter-enhancer (JCV(L)). In this study, a JCV Pnt2 oligonucleotide probe was used to screen a cDNA expression library from glial P19 mouse embryonal carcinoma cells. A cDNA clone was isolated by Southwestern blot assay and it produced a protein that reproducibly and specifically bound to Pnt2. This cDNA had 100% homology to one of three previously identified mouse cDNAs called cellular nucleic acid binding proteins (Cnbps). Cnbps are a highly homologous family of eukaryotic genes implicated in functional interactions with cytoplasmic RNA and regulatory DNA elements. An mRNA of 2.2 kb of Pnt2-interacting Cnbp (PCnbp) was seen in undifferentiated, muscle or glial P19 cells. When expressed from a cDNA expression vector as a fusion protein that also contained 115 kDa from beta-galactosidase, a Pnt2 binding protein (PCNBP) specifically bound to Pnt2 in Southwestern blots as a 30 kDa component of the 145 kDa fusion protein. Furthermore, JCV(E) expression was negatively regulated by PCnbp produced in vivo from the cDNA expression vector. Regulation of JCV(L) was unaffected. We suggest a novel role for CNBP as a PCNBP that interacts with Pnt2 in the negative transcriptional regulation of JCV(E).

Different HPV16 E6/E7 oncogene expression patterns in epithelia reconstructed from HPV16-immortalized human endocervical cells and genital keratinocytes.

Human papillomavirus type 16 (HPV16) E6/E7 oncogenes immortalize two types of human genital epithelial cells in vitro, endocervical cells and ectocervical or foreskin keratinocytes. Epithelia reconstructed in in vivo nude mouse implants or in vitro organotypic raft cultures from immortalized endocervical cells form higher grade dysplasia than those from keratinocytes. Here, we compared viral E6/E7 mRNA expression in immortalized cell lines of the three cell types using implants, rafts and in situ hybridization assays. Endocervical cells expressed E6/E7 throughout their reconstructed epithelia. In contrast, oncogenes were limited to basal cells for keratinocyte lower grade dysplasias. To study the role of the HPV16 promoter/enhancer in this repression in the upper layers of keratinocyte epithelia, new cell lines were established by immortalization with E6/E7 controlled by the SV40 promoter. The oncogenes were shown to be controlled from the SV40 elements after immortalization. Nevertheless, E6/E7 in the two cell types had the same cell-specific expression pattern as that controlled from the homologous HPV16 promoter. In addition, naturally occurring premalignant lesions having integrated HPV16 DNA expressed E6/E7 extensively in the high-grade dysplastic region of undifferentiated metaplasia. On the other hand, oncogene expression was restricted to lower layers in the lower grade dysplastic region of more mature differentiation. Our data suggest that keratinocytes have an inherent HPV16 promoter-nonspecific mechanism of repression. Apparently this mechanism, which can be acquired during maturation, is initially nonfunctional in in vitro and in vivo epithelia derived from metaplastic endocervical cells.

Expression of cellular genes in HPV16-immortalized and cigarette smoke condensate-transformed human endocervical cells.

We studied the molecular mechanism of successive multistep cervical carcinogenic progression with our previously established in vitro model system. This system was composed of primary human endocervical cells (HEN), two lines of HEN immortalized by HPV16 and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). The expression was examined of diverse cellular genes associated with oncogenesis and senescence, especially for cervical cancer. Consistent results were seen for the pairs of immortalized and malignantly transformed lines. Immortalization of HEN by HPV16 resulted in enhanced expression of H-ras, c-myc, B-myb, p53, p16INK4 and PCNA mRNA; enhanced expression of p16 and PCNA proteins; decreased expression of WAF1/p21/Cip1/Sid1 and fibronectin mRNA; and decreased p53 protein. On the other hand, the CSC-transformed counterparts of HPV16-immortalized cells had up-regulated levels of B-myb, p53 and WAF1 mRNA and p53 protein. Our results indicate that the differential activation or inactivation of multiple cellular genes is important for the immortalization, as well as the transformation, of human cervical cells. Further, we suggest that our in vitro model system is useful for investigating the molecular mechanism of multistep cervical carcinogenesis.

Effect of glucocorticoid hormones on viral gene expression, growth, and dysplastic differentiation in HPV16-immortalized ectocervical cells.

Steroid hormones are proposed to act as cofactors with human papillomaviruses (HPVs) in the etiology of cervical cancer. We and others reported that progesterone and glucocorticoid hormones induce the expression of HPV16 via three glucocorticoid response elements (GREs) in the viral regulatory region. Consensus GREs (GREcs) are useful in other systems for examining the effect of hormones after enhancing the response with mutated GREc constructs. Therefore, this study used human ectocervical cells (HEC) and HPV type 16 containing three GREcs to establish immortalized cells (HEC-16GREc). Northern blot assays showed that the level of viral E6-E7 oncogene RNA was increased by hormones substantially more in HEC-16GREc than in wild-type HPV16-immortalized human ectocervical cells (HEC-16). The saturation density and the hormone response of the growth rate were significantly higher for HEC-16GREc and the doubling was faster in the presence of hormone than for HEC-16. Although both were nontumorigenic, only HEC-16GREc showed anchorage-independent growth, which was dependent on hormone. Also, HEC-16GREc were more abnormal in their epithelium differentiation pattern in organotypic (raft) cultures. Furthermore, hormone-treated HEC-16GREc rafts showed more dysplastic features than hormone-treated HEC-16 rafts. These results suggest new features of the role of hormones: that enhanced expression of viral oncogenes in response to hormones apparently confers a greater risk for cervical cells containing HPV16. Further, HEC-16GREc could be ideal for studying hormone-dependent and -independent malignant transformation.

Identification and location of human papillomavirus type 16 antisense early promoter and characterisation of antisense RNA.

Antisense RNA sequences of various regions of human papillomavirus type 16 (HPV 16) were previously found in a number of cervical lesions, but the viral or cellular promoter has not been identified. HPV 16 E7 oncogene antisense transcripts expressed from an antisense promoter in viral DNA were found in the present study by RNase protection assays for total and cytoplasmic RNA. The antisense promoter for these transcripts was located within HPV 16 nt 4030-4230 by deletion analyses. The results also suggested that most of the antisense RNA was relatively short. The antisense promoter of HPV 16 was functional for expression of antisense RNA of a heterologous gene. Antisense-sense double-stranded E7 RNA was detected, and the sense RNA of this duplex was apparently inefficient for splicing or cleavage/poly(A) addition. These results show that HPV 16 can produce early region antisense RNA, which is from a promoter within a defined region of the viral genome. The possible importance of these transcripts for the regulation of episomal HPV 16 gene expression in infected and premalignant lesions and the possible importance of their deregulation for expression in malignant lesions are discussed.

Dual NF1-requiring effect of human neurotropic JC virus composite pentanucleotide repeat elements on early and late viral gene expression.

Human polyoma JC virus (JCV) is a largely brain cell-specific virus that is associated with AIDS. The neurotropism involves the various transcription factors interacting with their cognate sequences in the JCV early (JCVE) and late (JCVL) promoters-enhancer, but the mechanism is unclear. The JCV tandem AGGGA pentanucleotide double repeat element (Pnt2) and the abutting NF1 site form a composite Pnt2 (cPnt2). Here, we studied the roles of both sites of cPnt2 in the expression of JCVE and JCVL. JCVE activity was progressively increased in U-87 MG human glioblastoma cells following the site-specific mutation of Pnt2 in one and both 98-bp repeats. In contrast, the activity for JCVL was progressively reduced by mutating single and double Pnt2s. However, JCVE activity was unaffected by mutating both Pnt2s when both NF1 sites of cPnt2 were mutated. The activity of JCVL was also unaffected by double Pnt2 mutations after the NF1 sites were mutated. Differentiating P19 glial cells showed similar results. Next, Pnt2-interacting proteins were examined by two in vitro assays with a Pnt2 oligonucleotide. Mobility shift assays showed one Pnt2-protein complex for U-87 MG cell extracts and two for P19 glial cell extracts. However, similar results were obtained for glial, muscle, and undifferentiated P19 cells. In UV cross-linking assays, two Pnt2-binding proteins (Pnt2BPs) were detected. The results suggested that the effects on JCVE and JCVL expression of Pnt2BP and NF1 via cPnt2 are dual and require a glial cell-specific NF1 function.

Induction of p16 during immortalization by HPV 16 and 18 and not during malignant transformation.

The p16 (MTS1) tumour-suppressor gene is a cyclin-dependent kinase (cdk) inhibitor that decelerates the cell cycle by inactivating the cdks that phosphorylate the retinoblastoma tumour-suppressor gene (Rb) protein (pRb). In cervical cancers, pRb is inactivated by the HPV E7 oncoprotein or by mutations. The hypothesis of earlier reports was that the disruption of the p16/cdk-cyclin/Rb cascade is essential for malignant cervical transformation/carcinogenesis. We previously established in vitro model systems of cervical cancer representing four steps of oncogenic progression initiated by the two most common oncogenic HPVs in ectocervical and endocervical epithelial cells. This report used these systems to investigate the role of p16 in cervical cancers. A dramatic enhancement of the p16 RNA level was observed after immortalization by HPV 16 or 18. Furthermore, the p16 protein was newly observed following immortalization. However, no further changes were found for RNA or protein levels after serum selection or malignant transformation. For three cervical carcinoma cell lines, similar high levels of p16 expression were seen. Point mutations or homozygous deletions of p16 were not observed in the in vitro systems or in clinical specimens. These results suggest that the inactivation of the p16/cdk-cyclin/Rb cascade does not occur during malignant transformation but occurs during the immortalization by HPV in HPV-harbouring premalignant lesions, the in situ equivalent of immortalized cells. Also suggested is that p16 has no role in the specific malignant transformation step from immortal premalignant lesions during the carcinogenesis of HPV-initiated cervical cancers.

Up-regulation of hormone response of human papillomavirus type 16 expression and increased DNA-protein binding by consensus mutations of viral glucocorticoid response elements.

Human papillomaviruses (HPVs) and steroid hormones are linked to the development of cervical cancer. Studies from our laboratory and others showed that the steroid glucocorticoid and progesterone hormones activated the expression of HPV type 16. This activation was attributed to the specific interaction of the glucocorticoid receptor (GR) with the three glucocorticoid response elements (GREs) in the HPV16 regulatory region. In the present study, we first examined the glucocorticoid response mediated through the GREs, using GRE consensus (GREc) mutations and expression assays from a heterologous basal promoter. Both single and triple HPV16 GREc constructs increased expression in the presence of the dexamethasone glucocorticoid in HeLa cervical carcinoma cells and primary baby rat kidney epithelial cells, in comparison with the triple wild-type GREs. Further, the hormone increased significantly the expression of the viral E6-E7 oncogene mRNA from intact HPV in primary human ectocervical cells in in situ hybridization assays. Three in vitro assays of DNA-protein interaction with oligonucleotides and HeLa cell extracts showed a higher binding of protein to two of the HPV16 GREcs than to the wild-type GREs. This applied especially to the GRE containing an overlapping NF1 half site, that also had a greater differential induction by dexamethasone of expression in vivo. The NF1 site was mutated in the GREc that also was bound by unique, lower-mobility complexes in electrophoretic mobility shift assays. UV-crosslinking assays confirmed the increased binding and showed binding by a 96-kDa protein, probably the GR. Our results show an important role of glucocorticoids in HPV16 expression. The direct action through the HPV16 GREs is suggested to be mediated by the hormone-activated GR in association with other factors.

Human JC virus nuclear factor 1 binding motifs and large tumor antigen region rquired for transactivation of late promoter.

The nuclear factor 1 (NF-1) motifs, NF-1 II/III, In the two 98-bp repeats of the transcription-regulatory region of JC virus (JCV), have a critical role in brain-specific transcription from the JCV early promoter-enhancer. In this study, the role of these motifs in transactivation of the JCV late promoter-enhancer (JCVL) was examined in differentiating glial P19 embryonal carcinoma cells. The expression of papovaviral large tumor antigen (T-Ag) in the glial cells was shown by double immunofluorescence assays. By using site-directed mutagenesis and in vivo assays, the two wild-type NF-1 II/III sites, but not the third site, were found to be essential for the transactivation of JCVL by JCV T-Ag. In vitro transcription assays confirmed this specific transactivation and the transactivation was abolished by T-Ag antibody. In electrophoretic mobility shift assays, expression of JCV T-Ag increased the binding of a factor(s) to the 98-bp repeat. T-Ag antibody abolished the increase of binding. Binding assays with oligonucleotides of NF-1 11/111 motifs showed that the increased binding specifically required the wild-type NF-1 II/III sequences and confirmed the requirement of T-Ag. To determine the region of T-Ag necessary for transactivation Of JCVL, the coding sequences were mutated. The amino-terminal region of JCV Ag in amino acids 1-437 was essentially required for efficient transactivation. These results indicated that transactivation of JCVL and increased binding require a factor(s) found specifically in glial cells, the JCV NF-1 II/III sites, and the T-Ag amino-terminal region.

Glial and muscle embryonal carcinoma cell-specific independent regulation of expression of human JC virus early promoter by cyclic AMP response elements and adjacent nuclear factor 1 binding sites.

The human polyoma JC virus (JCV) is a glial cell-specific virus and is the etiological agent for the terminal AIDS-associated brain disease, progressive multifocal leukoencephalopathy (PML). JCV contains several binding sites for transcriptional factors that are important for activity in glial cells, including cyclic AMP (cAMP) response elements (CREs) which are four nucleotides from nuclear factor 1 (NF1) sites within the two 98 bp repeat regions. We studied the combined role of cAMP and NF1 in regulating the expression of the JCV early promoter-enhancer (JCVE) in differentiating glial and muscle P19 embryonal carcinoma cells. JCVE expression remained several-fold higher in the presence of cAMP in glial cells, irrespective of whether the relatively strong activity of JCVE was greatly reduced by NF1 site mutations. In contrast, cAMP had no effect in muscle cells, independent of whether the modest activity of JCVE was two-fold higher due to NF1 site mutations. The in vivo effects were confirmed with in vitro transcription assays using glial cell extracts, competitors of CRE, and the NF1 site, and single repeat JCVE region with mutations in the NF1 II/ III binding sites as templates. The in vitro results also indicated that the effects were due to the CREs of JCV, rather than to the indirect effects of cAMP. Overall, the results indicated that NF1 and cAMP have independent, different, tissue-specific, and direct effects in the regulation of JCVE. These effects may contribute the neurotropic PML-inducing pattern of expression of JCVE.

Evidence for a mechanism of demyelination by human JC virus: negative transcriptional regulation of RNA and protein levels from myelin basic protein gene by large tumor antigen in human glioblastoma cells.

Human JC virus (JCV) is a neurotropic human polyomavirus that was found in the plaques and oligodendroglial cells of the brains of patients with the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Transgenic mice expressing JCV large tumor (T)-antigen from integrated DNA showed dysmyelination in the central nervous system. However, the role of T-antigen from episomal DNA in the demyelination in PML remains unclear. In this report, we examined the effect of episomally expressed JCV T-antigen on the expression of myelin basic protein (MBP) in U-87 MG human glioblastoma cells to study the mechanism of demyelination. Expression assays of the MBP promoter in U-87 MG detected a 2.5-fold reduction in cells expressing intact T-antigen. Next, U-87 MG expressing T-antigen were examined by RNase protection assays for mRNA accumulation from the endogenous MBP promoter. Also, the expression of the MBP promoter plasmid was determined using in vitro transcription assays with extracts from T-antigen expressing cells. Both assays found a similar down-regulation of the MBP promoter by T-antigen, confirming that negative regulation occurred at the transcriptional level for the endogenous and exogenous MBP promoters. Furthermore, in situ immunofluorescence assays and quantitative Western blot analysis provided convincing evidence of a similar reduction in the level of MBP produced from the functional endogenous gene in U-87 MG glioblastoma cells expressing T-antigen. Thus, we provide evidence for the role of T-antigen in a transcriptional control mechanism for the demyelination that is caused by JCV in PML patients.

Identification of two novel cellular genes associated with multistage carcinogenesis of human endocervical cells by mRNA differential display.

Carcinogenesis originating from cervical cells has been recognized as a multistage process in which human papillomavirus (HPV) infection and cofactors, such as cigarette smoke, are frequently involved in the development of malignant cancer. However, the molecular mechanism underlying this process remains poorly understood. To identify the cellular genes involved in multistage cervical oncogenesis, we used the mRNA differential display method to analyze primary human endocervical cells (HEN), HPV 16-immortalized cells (HEN-16) and cigarette smoke condensate (CSC)-transformed cells (HEN-16T). Two cDNAs--PA4 and PA9--have been identified and isolated after comparing 8000 cDNAs from HEN, HEN-16 and HEN-16T. Northern blot analysis showed that PA4 was expressed 2- to 3-fold higher in HEN-16 and HEN-16T than in HEN, whereas PA9 was expressed uniquely in HEN. Moreover, the same patterns of PA4 and PA9 expression were found for a second line of HPV 16-immortalized endocervical cells and the corresponding transformed cells. An analysis of cDNA sequences showed that PA4 and PA9 had no homology to nucleotide sequences in Genbank. We suggest that immortalization, but not tumorigenesis, up-regulated a new oncogene, PA4, and down-regulated a new tumor suppressor gene, PA9. These results demonstrated the utility of the human endocervical cell model system and the mRNA differential display method for identifying the genes that may be involved in multistage cervical carcinogenesis.

Malignant transformation of human ectocervical cells immortalized by HPV 18: in vitro model of carcinogenesis by cigarette smoke.

In addition to the established role of human papillomaviruses (HPVs) in cervical cancer, smoking has been suggested to be an important cofactor. Previously, primary human ectocervical cells immortalized by HPV types 16 and 18 DNA did not form tumors on nude mice. Here, we derived a new line of HPV 18-immortalized ectocervical cells (HEC-18-1), which was also non-tumorigenic. To examine the role of cigarette smoking in the progression of cervical cancer initiated by HPV 18, we adapted these cells to growth in serum and high calcium and treated the cells with cigarette smoke condensate until tumorigenic cells (HEC-18-1C) were produced. Moderate and late passage serum-adapted untreated HEC-18-1 (HEC-18-1S) remained non-tumorigenic. A typical HEC-18-1C tumor was an invasive squamous cell carcinoma, from which we established a clonal line of cells (HEC-18-1CT). Although the physical state of HPV 18 was not affected by malignant transformation and the gene expression of HPV 18 was not affected by malignant transformation and the gene expression of HPV 18 was affected little, the differentiation of the epithelium derived in organotypic (raft) culture from HEC-18-1CT was altered dramatically. Moderate and late passage HEC-18-1 and HEC-18-1S were reconstructed into mild dysplasia in organotypic (raft) culture. On the other hand, the moderate passage malignantly transformed HEC-18-1CT displayed severe dysplasia/carcinoma in situ in raft culture. We describe here the first direct evidence of the role of cigarette smoke in the progression of HPV-initiated carcinogenesis using an in vitro model system.