Biomedical subjects

H Fan

Publications and source records attributed to H Fan.

At least 73 records · Page 4Linked to original sources

PubMed2000

The long terminal repeat of Jaagsiekte sheep retrovirus is preferentially active in differentiated epithelial cells of the lungs.

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a contagious bronchioloalveolar carcinoma of sheep known as sheep pulmonary adenomatosis (SPA; ovine pulmonary carcinoma). JSRV is unique among retroviruses because it transforms the alveolar type II cells and the nonciliated bronchiolar cells (Clara cells) of the lungs; these cells are where JSRV is specifically expressed in both naturally and experimentally SPA-affected sheep. In this study, we investigated the cell specificity of JSRV expression. By transient-transfection assays of 23 different cell lines with a reporter plasmid driven by the JSRV long terminal repeat (LTR), pJS21-luc, we found that the JSRV LTR is preferentially active in cell lines derived from type II pneumocytes and Clara cells (MLE-15 and mtCC1-2 mouse cell lines). Reporter assays using progressive 5' deletions of pJS21-luc allowed us to establish that the JSRV enhancers are able to activate the JSRV proximal promoter in MLE-15 and mtCC1-2 cells, but they have very low activity in mouse cells of other lineages (e.g., NIH 3T3). The JSRV enhancers are able to activate heterologous promoters in both MLE-15 and 3T3 cells, although optimal activity is achieved in MLE-15 cells only with the homologous JSRV promoter. Thus, JSRV cell-specific LTR activity appears to result from an interaction between the enhancer elements and the JSRV proximal promoter elements. By mutation analysis, we established that an upstream NF-kappaB-like element appears to be responsible for approximately 50% of the JSRV LTR transcriptional activity in MLE-15 cells. Electrophoretic mobility shift assays showed evidence of a factor(s) that binds to this sequence. Antibody supershift experiments indicated that the factor(s) is not related to NF-kappaB component p50 or p52. This factor also appeared to be present in cells that do not support a high level of JSRV expression. Finally the JSRV(21) LTR contains putative enhancer binding motifs for transcription factors such as hepatocyte nuclear factor 3 (HNF-3) that are involved in lung-specific gene expression. Cotransfection experiments demonstrated that exogenous HNF-3 is able to enhance the expression of pJS21-luc in NIH 3T3 cells, which normally show minimal enhancer activity for the JSRV LTR.

3T3 Cells↗

PubMed2000

Molecular cloning and functional analysis of three type D endogenous retroviruses of sheep reveal a different cell tropism from that of the highly related exogenous jaagsiekte sheep retrovirus.

Integrated into the sheep genome are 15 to 20 copies of type D endogenous loci that are highly related to two exogenous oncogenic viruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). The exogenous viruses cause infectious neoplasms of the respiratory tract in small ruminants. In this study, we molecularly cloned three intact type D endogenous retroviruses of sheep (enJS56A1, enJS5F16, and enJS59A1; collectively called enJRSVs) and analyzed their genomic structures, their phylogenies with respect to their exogenous counterparts, their capacity to form viral particles, and the expression specificities of their long terminal repeats (LTRs). In addition, the pattern of expression of enJSRVs in vivo was studied by in situ hybridization. All of the three enJSRV proviruses had open reading frames for at least one of the structural genes. In particular, enJS56A1 had open reading frames for all structural genes, but it could not assemble viral particles when highly expressed in human 293T cells. We localized the defect for viral assembly in the first two-thirds of the gag gene by making a series of chimeras between enJS56A1 and the exogenous infectious molecular clone JSRV(21). Phylogenetic analysis distinguished five ovine type D retroviruses: enJSRV groups A and B, ENTV, and two exogenous JSRV groups (African versus United Kingdom/North America isolates). Transient transfection assays indicated that the LTRs of the three enJSRVs were not preferentially active in differentiated lung epithelial cells. This suggests that the pulmonary tropic JSRV developed from a type D retrovirus that did not have lung specificity. Consistent with this, in situ hybridization of a panel of normal ovine tissues revealed high expression of enJSRV mRNA in the luminal epithelium and glandular epithelium of the uterus; lower expression was localized in the lamina propria of the gut and in the bronchiolar epithelium of the lungs.

Amino Acid Sequence↗

PubMed2000

Moloney murine leukemia virus-induced tumors show altered levels of proapoptotic and antiapoptotic proteins.

Moloney murine leukemia virus (M-MuLV) is a replication-competent, simple retrovirus that induces T-cell lymphomas when inoculated into neonatal mice. The tumor cells are typically derived from immature T cells. During preleukemic times, a marked decrease in thymic size is apparent in M-MuLV-inoculated mice. We previously demonstrated that this thymic regression is correlated with enhanced levels of thymocyte apoptosis (C. Bonzon and H. Fan, J. Virol. 73:2434-2441, 1999). In this study, we investigated the apoptotic state of M-MuLV-induced tumors. M-MuLV-induced tumors were screened for expression of the apoptotic proteins Fas and Bcl-2 by three-color flow cytometric analysis. Single-positive (SP; CD4(+) CD8(-) and CD4(-) CD8(+)) tumor cells generally displayed lower cell surface expression of Fas than SP thymocytes from uninoculated control mice. Double-positive (DP; CD4(+) CD8(+)) M-MuLV-induced tumor cells fell into two categories: those with normal high levels of Fas and those with low levels of Fas. Additionally, the vast majority of DP tumors showed elevated Bcl-2 levels. The DP tumor cells retaining normal/high Fas expression were capable of transducing an apoptotic signal upon anti-Fas engagement. In addition, DP and CD4(+) SP tumor populations displayed higher levels of Fas ligand than normal thymocytes with the same phenotypes. In contrast, CD8(+) SP and CD4(-) CD8(-) tumors did not show elevated Fas ligand expression. There was no significant correlation between Fas and Fas ligand expression in the DP tumors, suggesting that Fas Ligand expression was not the driving force behind Fas down-regulation. These results suggest that both the Fas death receptor and mitochondrial pathways of apoptotic death are active in M-MuLV-induced tumors and that they must be modulated to permit cell survival and tumor outgrowth.

3T3 Cells↗

PubMed2000

In vivo genomic footprinting of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat enhancer sequences in HTLV-1-infected human T-cell lines with different levels of Tax I activity.

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) enhances viral gene expression through sequences in the U3 region of the viral long terminal repeat. These sequences consist of three imperfect 21-bp repeats (TRE-1s) and a region between the promoter-central and promoter-proximal 21-bp repeats (TRE-2). The TRE-1s contain a core cyclic AMP response element (CRE) motif and can be bound by CREB, ATF-1, ATF-2, and other members of the CREB-ATF superfamily of transcription factors. Tax enhances CREB binding to TRE-1 in vitro, and it promotes dimerization of CREB as well as other bZIP proteins. Using ligation-mediated PCR on in vivo dimethyl sulfate-treated HTLV-1-infected cell lines MT-2 and MT-4, we have compiled a profile of protein occupancy in the HTLV-1 enhancer sequences in the presence of high (MT-2) and low (MT-4) levels of biologically active Tax I. The in vivo footprinting showed that all three TRE-1s were bound by protein(s), but only in MT-2 cells. In MT-2 cells, all TRE-1s showed strong protection of the G residues in the central CRE, and the footprints extended to differing degrees into the GC-rich flanking sequences. This indicated Tax I-dependent loading of transcription factors onto the HTLV-1 TRE-1s in vivo. In vivo footprinting on TRE-2 indicated that this region was bound by proteins regardless of the Tax I status of the cell line. However, the presence of Tax I increased the extent and altered the profile of proteins binding TRE-2 in vivo.

Base Sequence↗

PubMed2000

[Proliferative signal transmission mediated by EpoR in leukemic cell lines].

OBJECTIVE: To investigate the expression of erythropoietin receptor (EpoR) in leukemic cell lines and to clarify the mechanism of proliferative signal transmission in the leukemic cell line KOCL-33 mediated by EpoR. METHODS: Biotinylated Epo and flow cytometry were used for EpoR expression, (3)H-TdR incorporation for cell proliferation, immunoprecipitation and Western blot analysis for the tyrosine phosphorylation of signal transmission proteins. RESULTS: (1) All the cell lines tested expressed EpoR except for T-lymphoid cell lines. The positivity rates ranged from 18% to 99% with an average of 52%, and was no difference among T, B and non-lymphoid cells. (2) The cell proliferation was significantly enhanced in response to Epo in 7 of 9 cell lines tested. The stimulating effects of Epo did not correlate with the densities of EpoR expressed on the cells. (3) The proliferation of KOCL-33 cells was significantly enhanced in response to Epo. Dimerization and tyrosine phosphorylation of EpoR and tyrosine phosphorylation of JAK2 occurred in 1 min and tyrosine phosphorylation of STAT5 in 5 minutes after Epo stimulation. CONCLUSION: (1) There was EpoR expression in leukemic cell lines. (2) The cell proliferation could be stimulated by Epo in some leukemic cell lines. The stimulating effect of Epo did not correlate with the densities of EpoR expressed on the cells. (3) Dimerization and tyrosine phosphorylation of EpoR occurred, and JAK2, STAT5 involved in the proliferative signal transmission in KOCL-33 cells mediated by EpoR.

Cell Division↗

PubMed2000

[P15(INK4B) gene methylation in malignant hematopoietic diseases].

OBJECTIVE: To study the effect of operative region hypermethylation gene in human malignant hematopoietic tumors. METHODS: The abnormal methylation rate of P(15)(INK4B) gene 5'CpG island in 68 cases of malignant hematopoietic tumor samples were determined by methylation specific PCR using bisulfite modified DNA. RESULTS: The methylation rates of P(15)(INK4B) were 84%, 0, 50% and 75%, respectively, for 25 cases of acute myeloid leukemia (AML), 15 chronic myeloid leukemia (CML), 16 myelodysplastic syndrome (MDS) and 12 multiple myeloma (MM). P(15)(INK4B) gene was frequently methylated in patients with high risk MDS and early stage of MM. CONCLUSION: Hypermethylation of P(15)(INK4B) gene is one of the main causes of its inactivation. Hypermethylation of CpG island was closely related to the development of malignant hematopoietic diseases.

Acute Disease↗

PubMed2000

[Study of polyaluminium ferric chloride (PAFC) aqueous solutions visible spectra: the relationship among their absorbance at 460 nm, basicities and total Fe(III) concentration of PAFC solutions].

The visible spectra of forty-five synthesized polyaluminium ferric chloride (PAFC) aqueous solutions with different [OH]/[Al + Fe] (B) and nAl/nFe(molar ratio) were investigated. The results showed that their absorbances A of shoulder at 460 nm are all linear interrelationship with their B. We made the curve of absorbance A of PAFC with same B vs. nAl/nFe, there was a turning point. All of these points just located at the places of pH value rising higher in pH- nAl/nFe diagrams. For these reason, we thought that these points stand for the dynamic equilibrium of OH- between the polynucleus hydroxyl Fe(III) and Al(III). These points made certain relationship among the absorbance A at 460 nm, basicities and total Fe(III) concentration of PAFC solutions.

English Abstract↗

PubMed1999

Ectodomain shedding of TGF-alpha and other transmembrane proteins is induced by receptor tyrosine kinase activation and MAP kinase signaling cascades.

A variety of transmembrane proteins, such as transforming growth factor-alpha (TGF-alpha), tumor necrosis factor-alpha (TNF-alpha) and L-selectin, undergo shedding, i.e. cleavage of the ectodomain, resulting in release of a soluble protein. Although the physiological relevance of ectodomain shedding is well recognized, little is known about the signaling mechanisms activating this process. We show that growth factor activation of cell surface tyrosine kinase receptors induces ectodomain cleavage of transmembrane TGF-alpha through activation of the Erk MAP kinase signaling cascade without the need for new protein synthesis. In addition, expression of constitutively activated MEK1 or its downstream target Erk2 MAP kinase was sufficient to stimulate TGF-alpha shedding. The basal cleavage level in the absence of exogenous growth factor stimulation was due to p38 MAP kinase signaling. Accordingly, a constitutively activated MKK6, a p38 activator, activated TGF-alpha shedding in the absence of exogenous stimuli. In addition to TGF-alpha shedding, these mechanisms also mediate L-selectin and TNF-alpha cleavage. Thus, L-selectin shedding by neutrophils, induced by N-formylmethionyl-leucyl-phenylalanine, was strongly inhibited by inhibitors of Erk MAP kinase or p38 MAP kinase signaling. Our results indicate that activation of Erk and p38 signaling pathways may represent a general physiological mechanism to induce shedding of a variety of transmembrane proteins.

Animals↗

PubMed1999

Sonic hedgehog opposes epithelial cell cycle arrest.

Stratified epithelium displays an equilibrium between proliferation and cell cycle arrest, a balance that is disrupted in basal cell carcinoma (BCC). Sonic hedgehog (Shh) pathway activation appears sufficient to induce BCC, however, the way it does so is unknown. Shh-induced epidermal hyperplasia is accompanied by continued cell proliferation in normally growth arrested suprabasal cells in vivo. Shh-expressing cells fail to exit S and G2/M phases in response to calcium-induced differentiation and also resist exhaustion of replicative growth capacity. In addition, Shh blocks p21(CIP1/WAF1)-induced growth arrest. These data indicate that Shh promotes neoplasia by opposing normal stimuli for epithelial cell cycle arrest.

Animals↗

PubMed1999

Location of ryanodine receptor binding site on skeletal muscle triadin.

The binding between intact triadin or expressed triadin peptides and the ryanodine receptor has been investigated using membrane overlay and affinity chromatography. Ryanodine receptor binds to triadin blotted onto nitrocellulose with a KD of 40 nM in a medium containing 150 mM NaCl. The binding is substantially inhibited by hypertonic salt solution. Blot overlay experiments show that ryanodine receptor binds to bacterially expressed peptides, triadin(110-280), triadin(110-267), and triadin(279-674), but to no other moieties of the protein (numbers in parentheses are the residue positions). This binding is strongly inhibited by hypertonic salt solution. The same three triadin peptides as well as triadin(68-267), when attached to a glutathione column, bind to the ryanodine receptor. However, triadin(110-280) binds with high affinity, while triadin(68-267), triadin(110-267), and triadin(279-674) bind with low affinity. Triadin(258-280), triadin(267-280), and triadin(258-299) all bind to the ryanodine receptor with high affinity. On the other hand, a construct containing triadin(267-280), but preceded by nine residues of heterologous amino acids, does not bind significantly. These observations indicate two types of binding between triadin and the ryanodine receptor: (1) a low-affinity ionic interaction of large portions of triadin; (2) a specific high-affinity binding of a short relatively hydrophobic segment. The binding of this segment is probably the physiologically important domain for attachment between triadin and the ryanodine receptor.

Amino Acid Sequence↗

PubMed1999

Molecular and phylogenetic analysis of SRS 19-6 murine leukemia virus.

The complete nucleotide sequence of the genome of Solid-type Reticulum cell Sarcoma 19-6 murine leukemia virus (SRS 19-6 MuLV) was determined. This virus was isolated in mainland China from laboratory mice that had been separated from western mice since the 1930s. The genome is 8,256 nucleotides in length and exhibits a genetic organization characteristic of replication competent MuLVs. Phylogenies constructed from reverse transcriptase (RT) domains showed that SRS 19-6 MuLV is closely related to other MuLV-related retroviruses; however, it has clearly diverged from previously isolated MuLVs. Comparative sequence analysis of the env sequences indicated that SRS 19-6 MuLV encodes a surface (SU) glycoprotein that is related to other ecotropic MuLVs in the VR-A and VR-B variable regions. However, SRS 19-6 MuLV env glycoprotein was distinct from all other MuLVs (ecotropic and non-ecotropic) in the proline-rich hypervariable region. No evidence for recombination with endogenous MuLV env sequences in generation of SRS 19-6 MuLV was observed. Comparisons of long terminal repeat (LTR) sequences revealed that the GV 1.4 molecular clone of Graffi MuLV contained 96% sequence identity to SRS 19-6 MuLV's LTR with 99% identity when comparisons were restricted to the U3 regions of the two viruses. The consensus enhancer binding motifs contained in the U3 regions of the two viruses were nearly identical. Nevertheless the two viruses have previously been shown to induce distinct patterns of disease. Comparisons between 196 and Graffi GV1.4 MuLVs may provide insights into the mechanisms of disease specificity induced by MuLVs.

Amino Acid Sequence↗

PubMed1999

Immunization via hair follicles by topical application of naked DNA to normal skin.

In order to test the immune response generated to small amounts of foreign protein in skin, we applied naked DNA in aqueous solution to untreated normal skin. Topical application of plasmid expression vectors for lacZ and the hepatitis B surface antigen (HBsAg) to intact skin induced antigen-specific immune responses that displayed TH2 features. For HBsAg, specific antibody and cellular responses were induced to the same order of magnitude as those produced by intramuscular injection of the commercially available recombinant HBsAg polypeptide vaccine. Finally, topical gene transfer was dependent on the presence of normal hair follicles.

Administration, Topical↗

PubMed1999

In vitro infection of ovine cell lines by Jaagsiekte sheep retrovirus.

Sheep pulmonary adenomatosis (SPA), also known as jaagsiekte or ovine pulmonary carcinoma, is a contagious lung cancer of sheep, originating from type II pneumocytes and Clara cells. Previous studies have implicated a type D retrovirus (jaagsiekte sheep retrovirus [JSRV]) as the causative agent of SPA. We recently isolated a proviral clone of JSRV from an animal with a spontaneous case of SPA (JSRV(21)) and showed that it harbors an infectious and oncogenic virus. This demonstrated that JSRV is necessary and sufficient to induce SPA. A major impediment in research on JSRV has been the lack of an in vitro tissue culture system for the virus. The experiments reported here show the first successful in vitro infection with this virus, using the JSRV(21) clone. JSRV(21) virus was obtained by transiently transfecting human 293T cells with a plasmid containing the JSRV(21) provirus driven by the human cytomegalovirus immediate-early promoter. Virus produced in this manner exhibited reverse transcriptase (RT) activity that banded at 1.15 g/ml in sucrose density gradients. Infection of concentrated JSRV(21) into ovine choroid plexus (CP), testes (OAT-T3), turbinate (FLT), and intestinal carcinoma (ST6) cell lines resulted in establishment of infection as measured by PCR amplification. Evidence that this reflected genuine infection included the fact that heat inactivation of the virus eliminated it, the levels of viral DNA increased with passage of the infected cells, and the infected cells released active RT as measured by the sensitive product enhancement RT assay. The RT activity released from the infected cells banded at 1.15 g/ml, and JSRV(21) provirus was transmitted from infected cells to uninfected ones by cocultivation. However, the amount of virus released from infected cells was low. These results suggest that the JSRV receptor is present on many ovine cell types and that the observed restriction of JSRV expression in vivo to tumor cells might be controlled by factors other than the viral receptor. Finally we tagged the U3 of pJSRV(21) with the bacterial supF gene, an amber suppressor tRNA gene. The resulting clone, termed pJSRV(supF), is infectious in vitro. It may be a useful tool for future studies on viral DNA integration, since the normal sheep genome contains 15 to 20 copies of highly JSRV-related endogenous sequences that cross-react with many JSRV hybridization probes.

Animals↗

PubMed1999

Identification of directly infected cells in the bone marrow of neonatal moloney murine leukemia virus-infected mice by use of a moloney murine leukemia virus-based vector.

Early bone marrow infection of Moloney murine leukemia virus (M-MuLV)-infected mice was studied. Previous experiments indicated that early bone marrow infection is essential for the efficient development of T lymphoma. In order to identify the cellular pathway of infection in the bone marrow, infection of mice with a helper-free replication-defective M-MuLV-based retroviral vector was carried out. Such a vector will undergo only one round of infection, without spreading to other cells; thus, cells infected by the initially injected virus (directly infected cells) can be identified. For these experiments, the BAG vector that expresses bacterial beta-galactosidase was employed. Neonatal NIH/Swiss mice were inoculated intraperitoneally with ca. 10(6) infectious units of a BAG vector pseudotyped with M-MuLV proteins, and bone marrow cells were recovered 2 to 12 days postinfection. Single-cell suspensions were tested for infection by staining with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) or by immunofluorescence with an anti-beta-galactosidase antibody. Two sizes of infected cells were evident: large multinucleated cells and small nondescript (presumptively hematopoietic) cells. Secondary stains for lineage-specific markers indicated that the large cells were osteoclasts. Some of the small cells expressed nonspecific esterase, which placed them in the myeloid lineage, but they lacked markers for hematopoietic progenitors (mac-1, gr-1, sca-1, and CD34). These results provide evidence for primary M-MuLV infection of osteoclasts or osteoclast progenitors in the bone marrow, and they suggest that known hematopoietic progenitors are not primary targets for infection. However, the subsequent spread of infection to hematopoietic progenitors was indicated, since bone marrow from mice infected in parallel with replication-competent wild-type M-MuLV showed detectable infection in small cells positive for mac-1 or CD34, as well as in osteoclasts.

3T3 Cells↗

PubMed1999

Moloney murine leukemia virus-induced preleukemic thymic atrophy and enhanced thymocyte apoptosis correlate with disease pathogenicity.

Moloney murine leukemia virus (M-MuLV) is a replication-competent, simple retrovirus that induces T-cell lymphoma with a mean latency of 3 to 4 months. During the preleukemic period (4 to 10 weeks postinoculation) a marked decrease in thymic size is apparent for M-MuLV-inoculated mice in comparison to age-matched uninoculated mice. We were interested in studying whether the thymic regression was due to an increased rate of thymocyte apoptosis in the thymi of M-MuLV-inoculated mice. Neonatal NIH/Swiss mice were inoculated subcutaneously (s.c.) with wild-type M-MuLV (approximately 10(5) XC PFU). Mice were sacrificed at 4 to 11 weeks postinoculation. Thymic single-cell suspensions were prepared and tested for apoptosis by two-parameter flow cytometry. Indications of apoptosis included changes in cell size and staining with 7-aminoactinomycin D or annexin V. The levels of thymocyte apoptosis were significantly higher in M-MuLV-inoculated mice than in uninoculated control animals, and the levels of apoptosis were correlated with thymic atrophy. To test the relevance of enhanced thymocyte apoptosis to leukemogenesis, mice were inoculated with the Mo+PyF101 enhancer variant of M-MuLV. When inoculated intraperitoneally, a route that results in wild-type M-MuLV leukemogenesis, mice displayed levels of enhanced thymocyte apoptosis comparable to those seen with wild-type M-MuLV. However, in mice inoculated s.c., a route that results in attenuated leukemogenesis, significantly lower levels of apoptosis were observed. This supported a role for higher levels of thymocyte apoptosis in M-MuLV leukemogenesis. To examine the possible role of mink cell focus-forming (MCF) recombinant virus in raising levels of thymocyte apoptosis, MCF-specific focal immunofluorescence assays were performed on thymocytes from preleukemic mice inoculated with M-MuLV and Mo+PyF101 M-MuLV. The results indicated that infection of thymocytes by MCF virus recombinants is not required for the increased level of apoptosis and thymic atrophy.

Animals↗

PubMed1999

Moloney murine leukemia virus infects cells of the developing hair follicle after neonatal subcutaneous inoculation in mice.

The nature of Moloney murine leukemia virus (M-MuLV) infection after a subcutaneous (s.c.) inoculation was studied. We have previously shown that an enhancer variant of M-MuLV, Mo+PyF101 M-MuLV, is poorly leukemogenic when used to inoculate mice s.c., but not when inoculated intraperitoneally. This attenuation of leukemogenesis correlated with an inability of Mo+PyF101 M-MuLV to establish infection in the bone marrow of mice at early times postinfection. These results suggested that a cell type(s) is infected in the skin by wild-type but not Mo+PyF101 M-MuLV after s.c. inoculation and that this infection is important for the delivery of infection to the bone marrow, as well as for efficient leukemogenesis. To determine the nature of the cell types infected by M-MuLV and Mo+PyF101 M-MuLV in the skin after a s.c. inoculation, immunohistochemistry with an anti-M-MuLV CA antibody was performed. Cells of developing hair follicles, specifically cells of the outer root sheath (ORS), were extensively infected by M-MuLV after s.c. inoculation. The Mo+PyF101 M-MuLV variant also infected cells of the ORS but the level of infection was lower. By Western blot analysis, the level of infection in skin by Mo+PyF101 M-MuLV was approximately 4- to 10-fold less than that of wild-type M-MuLV. Similar results were seen when a mouse keratinocyte line was infected in vitro with both viruses. Cells of the ORS are a primary target of infection in vivo, since a replication defective M-MuLV-based vector expressing beta-galactosidase also infected these cells after a s.c. inoculation.

Animals↗

PubMed1999

Appearance of mink cell focus-inducing recombinants during in vivo infection by moloney murine leukemia virus (M-MuLV) or the Mo+PyF101 M-MuLV enhancer variant: implications for sites of generation and roles in leukemogenesis.

One hallmark of murine leukemia virus (MuLV) leukemogenesis in mice is the appearance of env gene recombinants known as mink cell focus-inducing (MCF) viruses. The site(s) of MCF recombinant generation in the animal during Moloney MuLV (M-MuLV) infection is unknown, and the exact roles of MCF viruses in disease induction remain unclear. Previous comparative studies between M-MuLV and an enhancer variant, Mo+PyF101 MuLV, suggested that MCF generation or early propagation might take place in the bone marrow under conditions of efficient leukemogenesis. Moreover, M-MuLV induces disease efficiently following both intraperitoneal (i.p.) and subcutaneous (s.c.) inoculation but leukemogenicity by Mo+PyF101 M-MuLV is efficient following i.p. inoculation but attenuated upon s. c. inoculation. Time course studies of MCF recombinant appearance in the bone marrow, spleen, and thymus of wild-type and Mo+PyF101 M-MuLV i.p.- and s.c.-inoculated mice were carried out by performing focal immunofluorescence assays. Both the route of inoculation and the presence of the PyF101 enhancer sequences affected the patterns of MCF generation or early propagation. The bone marrow was a likely site of MCF recombinant generation and/or early propagation following i.p. inoculation of M-MuLV. On the other hand, when the same virus was inoculated s.c., the primary site of MCF generation appeared to be the thymus. Also, when Mo+PyF101 M-MuLV was inoculated i.p., MCF generation appeared to occur primarily in the thymus. The time course studies indicated that MCF recombinants are not involved in preleukemic changes such as splenic hyperplasia. On the other hand, MCFs were detected in tumors from Mo+PyF101 M-MuLV s. c.-inoculated mice even though they were largely undetectable at preleukemic times. These results support a role for MCF recombinants late in disease induction.

Enhancer Elements, Genetic↗

PubMed1999

Jaagsiekte sheep retrovirus is necessary and sufficient to induce a contagious lung cancer in sheep.

Sheep pulmonary adenomatosis (SPA) is a contagious and experimentally transmissible lung cancer of sheep resembling human bronchiolo-alveolar carcinoma. A type D retrovirus, known as jaagsiekte sheep retrovirus (JSRV), has been associated with the etiology of SPA, but its exact role in the induction of the tumor has not been clear due to the lack of (i) a tissue culture system for the propagation of JSRV and (ii) an infectious JSRV molecular clone. To investigate the role of JSRV in the etiology of SPA, we isolated a full-length JSRV proviral clone, pJSRV21, from a tumor genomic DNA library derived from a natural case of SPA. pJSRV21 was completely sequenced and showed open reading frames in agreement with those deduced for the original South African strain of JSRV. In vivo transfection of three newborn lambs by intratracheal inoculation with pJSRV21 DNA complexed with cationic lipids showed that pJSRV21 is an infectious molecular clone. Viral DNA was detected in the peripheral blood mononuclear cells (PBMCs) of the transfected animals by a highly sensitive JSRV-U3 heminested PCR at various time points ranging from 2 weeks to 6 months posttransfection. In addition, proviral DNA was detected in the PBMCs, lungs, and mediastinal lymph nodes of two lambs sacrificed 9 months posttransfection, but no macroscopic or histological SPA lesion was induced. We prepared JSRV particles by transient transfection of 293T cells with a JSRV construct (pCMV2JS21) in which the upstream U3 was replaced with the cytomegalovirus early promoter. Four newborn lambs were inoculated with JSRV21 particles produced in this manner, and two of them showed the classical signs of SPA 4 months postinfection. The resulting tumors were positive for JSRV DNA and protein. Thus, JSRV21 is an infectious and pathogenic molecular clone and is necessary and sufficient to induce sheep pulmonary adenomatosis.

Animals↗