Persistence of the cytomegalovirus genome in human cells.

A small percentage of human fibroblast cells survived high-multiplicity infection by cytomegalovirus and were isolated as persistently infected cultures. Approximately 30% of the cells were in the productive phase of infection, since virus-specific structural antigens and virions were associated with these cells. The remaining cells contained neither viral structural antigens nor particles. Nuclear DNA from these nonproductive cells contained approximately 120 genome equivalents of viral DNA per cell as determined by reassociation kinetics. In situ hybridization confirmed that nuclei from nonproductive cells contained a significant amount of viral DNA that was distributed in most of these cells. Early virus-induced proteins and antigens were also detected. Nonproductive cells continued to grow, and there was a slow, spontaneous transition of some of these cells to productive viral replication. The majority of the viral DNA in nonproductive cells persisted with restricted gene expression. When infectious virus production was eliminated by growing the persistently infected cultures in the presence of anticytomegalovirus serum, approximately 45 genome equivalents of the viral DNA persisted per cell. The reassociation reaction approached completion. After removal of the antiserum and subculturing, infectious virus production resumed. Therefore, it was assumed that all sequences of the viral genome remained associated with these cells. Restriction of cytomegalovirus gene expression in persistently infected cell cultures is discussed.     

Presence of human papillomavirus genome in human tumor cell lines and cultured cells.

A series of 47 human carcinoma cell lines and their cultured cells were examined for human papillomavirus (HPV) genomes with the use of an HPV detection kit (DNA-RNA hybridization, mixed HPV DNA probe of types 6, 11, 16, 18, 31, 33 and 35). Four of 8 cases of mild dysplasia, 3 of 9 cases of severe dysplasia, 3 of 7 cases of carcinoma in situ, 3 of 15 cases of uterine carcinoma and 5 of 6 cases of condyloma acuminatum were shown to contain the HPV DNA genome in primary cultured cells, while HPV was not detected in the third-passage cells except for the three cases of large cell, nonkeratinizing squamous cell carcinoma. HPV was also not detected in such normal tissues as uterine cervical squamous epithelium, uterine cervical columnar epithelium and endometrium. The presence of HPV DNA genomes was detected consistently in the passages of three lines (SKG-II, HKMUS and HKTUS; large cell nonkeratinizing squamous cell carcinomas of the uterine cervix) with the use of the Southern Blot method (DNA-DNA hybridization, mixed HPV probe of types 6, 11, 16 and 18). HPV type 16 DNA was detected in HKTUS, and HPV type 18 DNA was found in SKG-II and HKMUS. The other 44 cell lines, including ovarian carcinoma, endometrial carcinoma, sarcoma, gastric cancer, pancreatic cancer and rectal cancer, were negative for the HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35 genomes under stringent hybridization conditions.     

Steroid hormone regulation of prostatic acid phosphatase expression in cultured human prostatic carcinoma cells

We have investigated the modulation of prostatic acid phosphatase expression in the human prostatic cancer cell line LNCaP in response to the natural androgens testosterone and dihydrotestosterone, the female sex steroid estradiol and the synthetic androgen R1881 (methyltrienolone). Testosterone and dihydrotestosterone at 1 microgram/ml enhance the acid phosphatase synthesis by a factor of 3.5, while a hundred-fold lower concentration of the synthetic androgen R1881 induces an almost five-fold increase in the expression of this enzyme. The stimulation by all androgens tested and estradiol was dose-dependent. The synthetic glucocorticoid triamcinolone acetonide does not modulate the prostatic acid phosphatase expression in LNCaP cells, neither alone nor in combination with R1881.     

A novel mechanism for persistence of human cytomegalovirus in macrophages.

Human cytomegalovirus (HCMV) infection of monocyte-derived macrophages (MDM) results in delayed and nonlytic productive viral growth. During late stages of replication, infectious virus remains cell associated in cytoplasmic vacuoles. In order to understand HCMV survival and persistence in MDM, we examined mechanisms involved in the formation and trafficking of HCMV-containing vacuoles in these cells. Utilizing double-label immunofluorescence with antibodies to viral and cellular proteins, HCMV-containing vacuoles were associated with the Golgi apparatus marker mannosidase II but not with markers to early endosomes (transferrin receptor and rab5) or late endosomes and early lysosomes (LAMP-1 and -2). In addition, as late-stage viral infection progressed in MDM, the cells displayed increasing abnormalities in the Golgi apparatus. Analysis of structural features of infected cells revealed the disruption of the microtubule network. These observations suggest a novel mechanism by which HCMV is vacuolized in MDM, avoiding degradation and release from the cell.     

The DNA sequence of the human cytomegalovirus genome.

In the first part of this article we review what has been learnt from the analysis of the sequence of HCMV. A summary of this information is presented in the form of an updated map of the viral genome. HCMV is representative of a major lineage of herpesviruses distinct from previously sequenced members of this viral family and demonstrates striking differences in genetic content and organization. The virus encodes approximately 200 genes, including nine gene families, a large number of glycoprotein genes, and homologues of the human HLA class I and G protein-coupled receptor genes. The HCMV sequence thus provides a sound basis for future molecular studies of this highly complex eukaryotic virus. The second part discusses the practical rate of DNA sequencing as deduced from this and other studies. The 229 kilobase pair DNA genome of human cytomegalovirus (HCMV) strain AD169 is the largest contiguous sequence determined to date, and as such provides a realistic benchmark for assessing the practical rate of DNA sequencing as opposed to theoretical calculations which are usually much greater. The sequence was determined manually and we assess the impact of new developments in DNA sequencing.     

Testosterone- and phorbol ester-stimulated proliferation in human cultured prostatic stromal cells

Prostatic stromal proliferation may be commonly associated with the development of benign prostatic hyperplasia. In this study, we investigate the role of testosterone and protein kinase C in stimulating cultured stromal cell proliferation. Testosterone increased the uptake of [(3)H]-thymidine into the human cultured prostatic stromal cells, this was reduced by the protein kinase C inhibitors, bisindolylymaleimide (10 nM) and myristoylated protein kinase C inhibitor (mPKCi, 20 microM), but not by G? 6983 (1 microM) or G? 6976 (1 microM). Cells responded to the addition of the PKC activators phorbol 12,13 dibutyrate (PDB), phorbol 12,13 diacetate (PDA), 12-deoxyphorbol 13-acetate (DPA) and 12-deoxyphorbol 13-tetradecanoate (DPT) with proliferation (order of potency DPT> or =PDB>PDA=DPA). The DPT-stimulated proliferative response was inhibited after cells were electroporated with PKCalpha antisense, but not mismatch oligonucleotides (8 microM). These results indicate that PKCalpha is involved in the proliferative response of human cultured prostatic stromal cells.     

Long-term effects of gamma-irradiation on cultured human thyroid cells

When differentiated 15-day-old cultures of human thyroid glands were irradiated they exhibited a high degree of radiosensitivity, an absence of split-dose recovery, an increase in the number of non-senescent colonies observed over four passages, increased focus formation on the confluent postirradiation monolayer and a shift in the isozyme pattern of LDH towards the anaerobic form (LD 5). The effects are similar to those previously observed for irradiated sheep thyroid cultures, but occurred at lower radiation doses.     

Functional role of PKC in contraction of cultured human prostatic stromal cells

The contractile activity of prostatic stromal cells contributes to symptoms of benign prostatic hyperplasia (BPH). However, the mechanisms for this contraction have not yet been fully elucidated. In this study, we investigated the role of protein kinase C (PKC) in prostatic contraction by measuring the isometric tension development of cultured human prostatic stromal cells (CHPSCs) derived from BPH patients. Fresh human BPH tissue was used only in a Western blot analysis. A ring preparation made of CHPSCs and collagen gel could develop an isometric tension during activation with various agonists. Phorbol 12,13 dibutyrate (PDBu), a PKC activator, induced a relaxation. A Western blot analysis revealed the expression of PKC-potentiated protein phosphatase-1 inhibitory protein (CPI-17) in both CHPSCs and fresh human BPH tissue to be much lower than that in the rabbit aorta. When CPI-17 was over-expressed, PDBu induced a large contraction, but the agonist-induced contraction did not become larger than expected. In alpha-toxin permeabilized preparations, PDBu induced a relaxation in control CHPSCs, while it induced a contraction at a constant [Ca2+]i in CPI-17 over-expressing CHPSCs. These results indicated that the activation of PKC in CHPSCs induces a relaxation probably due to low expression level of CPI-17 and also that the PKC-CPI-17 pathway does not appear to play a major role in the agonist-induced contraction even when CPI-17 was over-expressed.     

Infection of human cytomegalovirus in cultured human gingival tissue

Background

Matrix protein 2 (M2) is an integral tetrameric membrane protein of influenza A virus (IAV). Its ectodomain (M2e) shows remarkably little diversity amongst human IAV strains. As M2e-specific antibodies (Abs) have been shown to reduce the severity of infection in animals, M2e is being studied for its capability of providing protection against a broad range of IAV strains. Presently, there is little information about the concentration of M2e-specific Abs in humans. Two previous studies made use of ELISA and Western blot against M2e peptides and recombinant M2 protein as immunosorbents, respectively, and reported Ab titers to be low or undetectable. An important caveat is that these assays may not have detected all Abs capable of binding to native tetrameric M2e. Therefore, we developed an assay likely to detect all M2e tetramer-specific Abs.

Results

We generated a HeLa cell line that expressed full length tetrameric M2 (HeLa-M2) or empty vector (HeLa-C10) under the control of the tetracycline response element. These cell lines were then used in parallel as immunosorbents in ELISA. The assay was standardized and M2e-specific Ab titers quantified by means of purified murine or chimeric (mouse variable regions, human constant regions) M2e-specific Abs in the analysis of mouse and human sera, respectively. We found that the cell-based ELISA was substantially more effective than immobilized M2e peptide in detecting M2e-specific Abs in sera of mice that had recovered from repetitive IAV infections. Still, titers remained low (< 5 μg/ml) even after two consecutive infections but increased to ~50 μg/ml after the third infection. Competition with free M2e peptide indicated that ~20% of M2e-specific Abs engendered by infection reacted with M2e peptide. In humans presenting with naturally acquired influenza virus infection, 11 of 24 paired sera showed a ≥ 4-fold increase in M2e-specific Ab titer. The Ab response appeared to be of short duration as titers were very low (average 0.2 μg/ml) in all patients at onset of infection and in controls, in spite of evidence for previous exposure to IAV.

Conclusion

The results provide convincing evidence that M2e-specific Ab-mediated protection is currently lacking or suboptimal in humans.     

Adenovirus type 12-induced rat tumor cells of neuroepithelial origin: persistence and expression of the viral genome.

Four cell lines derived from adenovirus type 12-induced rat brain tumors were studied. The polyploid cells displayed neuroepithelial characteristics and were transplantable into syngeneic rats and nude mice. In tissue culture the cells grew in monolayers and multilayers. A very high saturation density was reached, and the cells plated in agar and were easily agglutinated with low concentrations of concanavalin A. Between 2 and 11 copies of the viral genome per diploid cellular genome were detected by reassociation kinetics analysis in the different lines. The patterns of distribution of viral DNA sequences in these lines, as revealed by blot analysis, suggest colinear integration of the intact viral genome into the cellular DNA. The patterns of integration were stable after more than 15 months of prolonged tissue culture and after animal reimplantation. Integration patterns were identical in three of the tumor lines and different in another line. Viral sequences were transcribed. The extent of homology found toward adenovirus type 12 DNA in polyadenylated polysome-associated mRNA isolated from the tumor lines suggests that the early and some of the late genes of adenovirus type 12 DNA are transcribed in these tumor cells. Infectious virus was not rescuable from these lines.     

Low persistence of radiation-induced centromere positive and negative micronuclei in cultured human cells

The micronucleus (MN) assay is widely used both in genetic toxicology and in the biomonitoring of human populations. Lymphocytes, cell lines, and bone marrow and epithelial cells are usually employed as target systems in such studies. However, little effort has been done to assess the persistence of MN in highly proliferative cells. To study the behaviour of MN containing whole chromosomes or acentric fragments, we have performed a time course experiment on the persistence of γ-ray (3 Gy) induced MN in a human lymphoblastoid cell line. The frequency and content of MN were analyzed 1, 3, 7, 14, and 56 days after irradiation by pancentromeric fluorescence in situ hybridization (FISH). We observed a clear induction of both centromere positive and negative MN at completion of the first mitotic division. The frequency of both types of MN drastically declined to basal levels 7 days after irradiation with an identical kinetics. We therefore conclude that centromere positive and negative MN are highly unstable upon cell division, indicating that the MN assay could not be a good biomarker of DNA damage induced by acute treatments in highly proliferative cells. The implication of our findings in biomonitoring and in genotoxicity studies is discussed.     

Low persistence of radiation-induced centromere positive and negative micronuclei in cultured human cells

The micronucleus (MN) assay is widely used both in genetic toxicology and in the biomonitoring of human populations. Lymphocytes, cell lines, and bone marrow and epithelial cells are usually employed as target systems in such studies. However, little effort has been done to assess the persistence of MN in highly proliferative cells. To study the behaviour of MN containing whole chromosomes or acentric fragments, we have performed a time course experiment on the persistence of gamma-ray (3 Gy) induced MN in a human lymphoblastoid cell line. The frequency and content of MN were analyzed 1, 3, 7, 14, and 56 days after irradiation by pancentromeric fluorescence in situ hybridization (FISH). We observed a clear induction of both centromere positive and negative MN at completion of the first mitotic division. The frequency of both types of MN drastically declined to basal levels 7 days after irradiation with an identical kinetics. We therefore conclude that centromere positive and negative MN are highly unstable upon cell division, indicating that the MN assay could not be a good biomarker of DNA damage induced by acute treatments in highly proliferative cells. The implication of our findings in biomonitoring and in genotoxicity studies is discussed.     

Protein kinase G II-mediated proliferative effects in human cultured prostatic stromal cells

This study investigates the effect of protein kinase G (PKG) activation upon proliferation of human cultured prostatic stromal cells. The PKG II activator (8-pCPT-cGMP; IC50 of 113+/-42 nM) and the phosphodiesterase inhibitor, zaprinast (up to 50 microM), but not the PKG I isoform activators (APT-cGMP and PET-cGMP), reduced foetal calf serum-stimulated proliferation. The effect of 8-pCPT-cGMP (30 microM) was blocked by Rp-8-Br-cGMPS (5 microM) and Rp-8-pCPT-cGMP (5 microM), but not Rp-cAMPS (5 microM). 8-pCPT-cGMP (30 microM) and zaprinast (50 microM), but not PET-cGMP (30 microM), caused a significant increase in atypical nuclei and an increase in annexin-V staining. These data indicate that activation of PKG II induces apoptosis of human cultured prostatic stromal cells.     

Long-term infection and shedding of human cytomegalovirus in T98G glioblastoma cells

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects, affecting primarily the central nervous system (CNS). To further understand this CNS pathology, cells from glioblastoma cell lines T98G and A172, the astrocytic glioblastoma cell line CCF-STTG1 (CCF), and the neuroblastoma cell line SH-SY5Y (SY5Y) were infected with HCMV. CCF and SY5Y cells were fully permissive for infection, while A172 cells were nonpermissive. In T98G cells, the majority of cells showed viral deposition into the nucleus by 6 h postinfection (hpi); however, viral immediate-early gene expression was observed in only approximately 30% of cells in the first 72 h. In viral antigen (Ag)-positive cells, although the development of complete viral replication centers was delayed, fully developed centers formed by 96 hpi. Interestingly, even at very late times postinfection, a mixture of multiple small, bipolar, and large foci was always present. The initial trafficking of input pp65 into the nucleus was also delayed. Titer and infectious-center assays showed a small number of T98G cells shedding virus at very low levels. Surprisingly, both Ag-positive and Ag-negative cells continued to divide; because of this continuous division, we adopted a protocol for passaging the T98G cells every third day to prevent overcrowding. Under this protocol, detectable infectious-virus shedding continued until passage 5 and viral gene expression continued through eight passages. This evidence points to T98G cells as a promising model for long-term infections.     

Characterization of cultured human prostatic epithelial cells by cluster designation antigen expression

Cultured prostatic epithelial cells have been extensively studied as a model of prostate biology. What is the lineage relationship of the cultured cells to the epithelial cell types in tissue? How different are cultured cells derived from tumor tissue to those derived from benign tissue? Expression of cluster designation (CD) cell surface molecules has been shown to be useful in characterizing cells according to lineage. A CD profile was therefore generated for cultured human prostatic epithelial cells and compared with those previously established for basal and luminal epithelial cells in the prostate. Presence of CD44, CD49b, CD49f, and CD104 and absence of CD57 suggests that cultured cells were derived from basal cells of prostatic tissues. However, expression of certain CD antigens characteristic of luminal epithelial cells was also observed in subpopulations of cultured cells. The pattern of CD antigens in cultured cells reflects a phenotype similar to that of transit-amplifying cells that have been described in the prostate. Several CD antigens were found expressed by both cultured prostatic epithelial and stromal cells, and are probably associated with cell proliferation. The CD profiles of cultured epithelial cell strains derived from normal compared with malignant tissues were notably similar to each other and to that of the prostate cancer cell line PC-3. We conclude that cells in culture retain expression of certain lineage-characteristic CD antigens. Furthermore, CD antigens can define subpopulations of cells with differential gene expression.     

Long-term cultured human neural stem cells undergo spontaneous transformation to tumor-initiating cells

In this report, we describe the spontaneous malignant transformation of long-term cultured human fetal striatum neural stem cells (hsNSCs, passage 17). After subcutaneous transplantation of long-term cultured hsNSCs into immunodeficient nude mice, 2 out of 15 mice formed xenografts which expressed neuroendocrine tumor markers CgA and NSE. T1 cells, a cell line that we derived from one of the two subcutaneous xenografts, have undergone continuous expansion in vitro. These T1 cells showed stem cell-like features and expressed neural stem cell markers nestin and CD133. The T1 cells were involved in abnormal karyotype, genomic instability and fast proliferation. Importantly, after long-term in vitro culture, the T1 cells did not result in subcutaneous xenografts, but induced intracranial tumor formation, indicating that they adjusted themselves to the intracranial microenvironment. We further found that the T1 cells exhibited an overexpressed level of EGFR, and the CD133 positive T1 cells showed a truncation mutation in the exons 2-7 of the EGFR (EGFRvIII) gene. These results suggest that continuous expansion of neural stem cells in culture may lead to malignant spontaneous transformation. This phenomenon may be functionally related to EGFR by EGFRvIII gene mutation.     

Cloning of the complete human cytomegalovirus genome in cosmids

Purified virion DNA (155 X 10(6) Mr) of human cytomegalovirus (CMV) strain Ad169 was partially cleaved with restriction endonucleases HindIII and EcoRI and cloned in the respective cleavage sites of cosmid pHC79. A complete gene library was established in a set of clones containing the viral DNA in long overlapping segments. Restriction maps for HindIII (29 fragments) and EcoRI (36 fragments) were constructed from the linkage of cosmid-cloned fragments, from double digestions of cloned DNA, and from blot hybridization of labeled cloned viral DNA with restriction fragments of virion DNA and singly or doubly cleaved cosmid clones.     

Human cytomegalovirus persistence and latency in endothelial cells and macrophages

Human cytomegalovirus (HCMV) is a clinically significant herpes virus that maintains a lifelong infection in the host. HCMV infection of endothelial cells and macrophages plays an important role in the establishment of latency and persistence, which appears critical for the maintenance of HCMV within the host. HCMV infection is profoundly influenced by endothelial cell origin and the specific pathway of macrophage differentiation. Multiple HCMV genes appear to be involved in enabling virus replication in these two cell types. Although the specific HCMV gene(s) mediating endothelial and macrophage tropism are unclear, a number of genetic determinants required for replication in these two cell types have been identified in the closely related murine cytomegalovirus (MCMV) mouse model, revealing novel mechanisms of virus tropism. This review focuses on recent advances in the understanding of HCMV replication in endothelial cells and macrophages, and the viral determinants that mediate replication in these two important cell types.