Roles of the Ras-MEK-mitogen-activated protein kinase and phosphatidylinositol 3-kinase-Akt-mTOR pathways in Jaagsiekte sheep retrovirus-induced transformation of rodent fibroblast and epithelial cell lines

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer of sheep. The virus can induce tumors rapidly, and we previously found that the JSRV envelope protein (Env) functions as an oncogene, because it can transform mammalian and avian fibroblast cell lines. (N. Maeda, Proc. Natl. Acad. Sci. USA 98:4449-4454, 2001). The molecular mechanisms of JSRV Env transformation are of considerable interest. Several reports suggested that the phosphatidylinositol 3-kinase/Akt pathway is important for transformation of mammalian fibroblasts but not for chicken fibroblasts. In this study, we found that Akt/mTOR is involved in JSRV transformation of mouse NIH 3T3 fibroblasts, because treatment with the mTOR inhibitor rapamycin reduced transformation. We also found that H/N-Ras inhibitor FTI-277 and MEK1/2 inhibitors PD98059 and U0126 strongly inhibited JSRV transformation of NIH 3T3 fibroblasts, suggesting that the H/N-Ras-MEK-mitogen-activated protein kinase (MAPK) p44/42 pathway is necessary for the transformation. In RK3E epithelial cells, the MEK1/2 inhibitors also eliminated transformation, but FTI-277 only partially inhibited transformation. It was noteworthy that p38 MAPK inhibitors enhanced JSRV transformation in both fibroblasts and epithelial cells. Treatment of transformed cells with p38 inhibitors both increased levels of phospho-MEK1/2 and phospho-p44/42 and induced rapid enhancement of the transformed phenotype. Immunohistochemical staining of tumor tissues from naturally and experimentally induced OPA and naturally occurring enzootic nasal adenocarcinoma revealed strong activation of MAPK p44/42 in all cases examined. However, p38 activation was not generally observed. These results indicate that signaling through two pathways (in particular, H/N-Ras-MEK-MAPK and, to a lesser extent, Akt-mTOR) is important for JSRV-induced transformation and that p38 MAPK has a negative regulatory effect on transformation, perhaps via MEK1/2 and p44/42.     

Expression of the jaagsiekte sheep retrovirus envelope glycoprotein is sufficient to induce lung tumors in sheep

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA). The expression of the JSRV envelope (Env) alone is sufficient to transform a variety of cell lines in vitro and induce lung cancer in immunodeficient mice. In order to determine the role of the JSRV Env in OPA tumorigenesis in sheep, we derived a JSRV replication-defective virus (JS-RD) which expresses env under the control of its own long terminal repeat (LTR). JS-RD was produced by transiently transfecting 293T cells with a two plasmid system, involving (i) a packaging plasmid, with the putative JSRV packaging signal deleted, expressing the structural and enzymatic proteins Gag, Pro, and Pol, and (ii) a plasmid which expresses env in trans for JS-RD particles and provides the genomes necessary to deliver JSRV env upon infection. During the optimization of the JS-RD system we determined that both R-U5 (in the viral 5' LTR) and the env region are important for JSRV particle production. Two independent experimental transmission studies were carried out with newborn lambs. Four of five lambs inoculated with JS-RD showed OPA lesions in the lungs at various times between 4 and 12 months postinoculation. Abundant expression of JSRV Env was detected in tumor cells of JS-RD-infected animals and PCR assays confirmed the presence of the deleted JS-RD genome. These data strongly suggest that the JSRV Env functions as a dominant oncoprotein in the natural immunocompetent host and that JSRV can induce OPA in the absence of viral spread.     

Envelope-induced cell transformation by ovine betaretroviruses

Ovine betaretroviruses include Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). JSRV and ENTV represent a unique class of oncogenic retroviruses that induce tumors of the respiratory tract. JSRV and ENTV are highly related but induce different diseases. Expression of the JSRV envelope (Env) induces transformation of rodent fibroblasts in vitro and phosphorylation of Akt, a central player in the phosphatidylinositol 3-kinase (PI-3K)/Akt signal transduction pathway. However, little information is available on the molecular biology of ENTV. In this study, we initially assessed whether the ENTV Env has the same properties as the homologous JSRV protein. We performed entry and interference assays using retroviral vectors pseudotyped with either the JSRV or the ENTV Env and sheep choroid plexus cells, choroid plexus cells stably expressing the JSRV Env protein, human 293T cells, mouse NIH 3T3 cells, or NIH 3T3 cells expressing human hyaluronidase 2 (HYAL2), the cellular receptor for JSRV. The results obtained indicated that ENTV and JSRV share the same receptor in sheep cells and that they can use human HYAL2 as a cellular receptor in mouse cells. The ENTV Env induces transformation of rodent fibroblasts in vitro. As with the JSRV Env, the tyrosine at position 590 is critical for ENTV Env-induced cell transformation, and Akt is phosphorylated in ENTV Env-transformed cells but not in the parental cell lines. Thus, ovine betaretroviruses share a common mechanism of cell transformation. We further investigated the relevance of Akt activation in cells transformed by ovine betaretroviruses. A PI-3K inhibitor blocked Akt phosphorylation in JSRV Env-transformed cells, suggesting a possible involvement of PI-3K in JSRV and ENTV Env-induced cell transformation. In addition, phosphorylated Akt was detected in a cell line derived from a lung tumor of a sheep with naturally occurring ovine pulmonary adenocarcinoma.     

Mutational analysis of the cytoplasmic tail of jaagsiekte sheep retrovirus envelope protein

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a transmissible lung cancer in sheep, ovine pulmonary adenocarcinoma. JSRV is unique in that the envelope protein functions as an oncogene, since it can morphologically transform fibroblast and epithelial cells in culture and can induce lung tumors in mice. Previous studies indicated that the transmembrane (TM) protein is essential for transformation, and particular attention has focused on a YXXM motif in the cytoplasmic tail. In this study, we carried out systematic mutagenesis of the cytoplasmic tail of JSRV Env. Alanine scanning mutagenesis revealed four classes of mutants: mutants in which transformation was abrogated, those in which transformation was not affected, those with reduced transformation, and those with increased transformation (supertransformers). In general, the alanine mutations did not affect Env protein production or its localization to the plasma membrane. Three functional domains of the cytoplasmic tail were identified: an amphipathic helix at the N-terminal (juxtamembrane) side, a nonessential C-terminal region, and an internal region (including the YXXM motif) where mutations resulted in abrogation, decreases, or increases in transformation. Alanine mutations in the amphipathic helix in both the hydrophobic and hydrophilic faces generally abolished transformation. The mutation R591A showed partial transformation that was consistent with loss of signaling through the Akt-mTOR pathway and signaling predominantly through the Ras-Raf-MEK1/2-extracellular signal-regulated kinase 1/2 pathway. The supertransforming mutants generally showed increased signaling through Akt and reduced activation of p38 MAPK that is inhibitory for transformation. These mutants provide further insight into the role of the TM cytoplasmic tail in JSRV transformation.     

Transformation of rodent fibroblasts by the jaagsiekte sheep retrovirus envelope is receptor independent and does not require the surface domain

Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of a contagious lung cancer of sheep known as ovine pulmonary adenocarcinoma (OPA). Expression of the JSRV envelope protein (Env) is sufficient to transform immortalized and primary fibroblasts, but the precise mechanisms of this process are not known. The cellular receptor for JSRV is hyaluronidase 2 (Hyal-2), the product of a putative tumor suppressor gene that in humans maps to a chromosomal region frequently deleted in the development of lung and breast cancers. Here we report studies to determine whether the Hyal-2-JSRV Env interaction plays a role in virus-induced transformation of rodent fibroblasts. Chimeric Env proteins between JSRV and the unrelated murine retroviruses Moloney murine leukemia virus (MMuLV) and mouse mammary tumor virus (MMTV) showed cell surface expression comparable to that of wild-type MMuLV Env and rescued infection of MMuLV particle pseudotypes. Interestingly, an MMuLV-JSRV chimera in which the putative receptor binding domain (RBD) and proline-rich region (PRR) of JSRV Env were replaced by the RBD and PRR of MMuLV induced transformation of 208F, a rodent fibroblast line. Cell lines derived from foci of MMuLV-JSRV chimera-transformed 208F cells grew in soft agar and showed Akt activation, a hallmark of JSRV-transformed rodent fibroblasts. Transformation assays performed using proteins with amino-terminal deletion mutations showed that the carboxy-terminal 141 amino acids of the transmembrane subunit (TM) were sufficient to induce cell transformation when targeted to the membrane with a myristoylation signal. Thus, the JSRV TM is necessary and sufficient to transform rodent fibroblasts. Taken together these results indicate that the interaction with Hyal-2 at least is not an essential determinant of JSRV-induced transformation of fibroblasts and that the viral TM functions essentially as an oncoprotein.     

Envelope proteins of Jaagsiekte sheep retrovirus and enzootic nasal tumor virus induce similar bronchioalveolar tumors in lungs of mice

Jaagsiekte sheep retrovirus (JSRV) induces bronchioalveolar tumors in sheep and goats. Expression of the JSRV envelope (Env) protein in mouse airway epithelial cells induces similar tumors, indicating that Env expression is sufficient for tissue-specific tumor formation. Enzootic nasal tumor virus (ENTV) is related to JSRV but induces tumors in the nasal epithelium of sheep and goats. Here we found that ENTV Env can also induce tumors in mice but, unexpectedly, with a phenotype identical to that of tumors induced by the JSRV Env, indicating that factors other than Env mediate the tissue specificity of tumor induction by ENTV.     

Role of virus receptor Hyal2 in oncogenic transformation of rodent fibroblasts by sheep betaretrovirus env proteins

The ovine betaretroviruses jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) cause contagious cancers in the lungs and upper airways of sheep and goats. Oncogenic transformation assays using mouse and rat fibroblasts have localized the transforming activity to the Env proteins encoded by these viruses, which require the putative lung and breast cancer tumor suppressor hyaluronidase 2 (Hyal2) to promote virus entry into cells. These results suggested the hypothesis that the JSRV and ENTV Env proteins cause cancer by inhibiting the tumor suppressor activity of Hyal2. Consistent with this hypothesis, we show that human Hyal2 and other Hyal2 orthologs that can promote virus entry, including rat Hyal2, can suppress transformation by the Env proteins of JSRV and ENTV. Furthermore, we provide direct evidence for binding of the surface (SU) region of JSRV Env to human and rat Hyal2. However, mouse Hyal2 did not mediate entry of virions bearing JSRV or ENTV Env proteins, bound JSRV SU poorly if at all, and did not suppress transformation by the JSRV or ENTV Env proteins, indicating that mouse Hyal2 plays no role in transformation of mouse fibroblasts and that the Env proteins can transform at least some cells by a Hyal2-independent mechanism. Expression of human Hyal2 in mouse cells expressing JSRV Env caused a marked reduction in Env protein levels, indicating that human Hyal2 suppresses Env-mediated transformation in mouse cells by increasing Env degradation rather than by exerting a more general Env-independent tumor suppressor activity.     

Multiple domains of the Jaagsiekte sheep retrovirus envelope protein are required for transformation of rodent fibroblasts

Jaagsiekte sheep retrovirus (JSRV) is an exogenous retrovirus of sheep that induces a contagious lung cancer, ovine pulmonary adenocarcinoma. We previously showed that the gene encoding JSRV envelope protein (Env) appears to function as an oncogene, since it can transform mouse NIH 3T3 cells. The cytoplasmic tail of the Env transmembrane protein (TM) is necessary for the transformation. However, previous experiments did not exclude the involvement of the Env surface protein (SU) in transformation. In this study, we created a series of nested deletion mutants through the SU domain and assessed their ability to transform rodent fibroblasts. All SU deletion mutants downstream of the predicted signal peptide were unable to transform murine NIH 3T3 or rat 208F cells. Transport to the plasma membrane of selected deleted Env proteins was confirmed by confocal immunofluorescence microscopy of hemagglutinin-tagged versions. Additional sequential SU deletion mutants lacking 50-amino-acid (aa) blocks throughout SU also were unable to transform. Furthermore, minimal insertion mutants of two amino acids (Leu/Gln) at various positions in SU also abolished transformation. These data indicate that domains in SU facilitate efficient JSRV transformation. This could reflect a necessity of SU for appropriate configuration of the Env protein or independent activation by SU of a signaling pathway necessary for transformation. Complementation between SU and TM mutants for transformation supported the latter hypothesis. Cotransfection with DeltaGP Y590F (mutant in the TM cytoplasmic tail) with DeltaGP SUDelta103-352 (lacking most of SU) resulted in efficient transformation. The resulting transformants showed evidence for the presence and expression of both mutant plasmids.     

Transformation of madin-darby canine kidney epithelial cells by sheep retrovirus envelope proteins

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) induce epithelial tumors in the airways of sheep and goats. In both of these simple retroviruses, the envelope (Env) protein is the active oncogene. Furthermore, JSRV Env can transform cultured cells by two distinct mechanisms. In rat and mouse fibroblasts, the cytoplasmic tail of JSRV Env is essential for transformation, which involves activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and the virus receptor hyaluronidase 2 (Hyal2) is not involved. In contrast, in the BEAS-2B human bronchial epithelial cell line, transformation is mediated by JSRV Env binding to Hyal2 followed by Hyal2 degradation and activation of the receptor tyrosine kinase RON, the activity of which is normally suppressed by Hyal2. Here we show that JSRV and ENTV Env proteins can also transform Madin-Darby canine kidney (MDCK) epithelial cells, but by a mechanism similar to that observed in fibroblast cell lines. In particular, the cytoplasmic tail of Env is required for transformation, the PI3K/Akt pathway is activated, expression of RON (which is not normally expressed in MDCK cells) does not affect transformation, and canine Hyal2 appears uninvolved. These results show that the JSRV and ENTV Env proteins can transform epithelial cells besides BEAS-2B cells and argue against a model for Env transformation involving different pathways that are uniquely active in fibroblasts or epithelial cells.     

Jaagsiekte sheep retrovirus infects multiple cell types in the ovine lung

Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The details of early events in the pathogenesis of OPA are not fully understood. For example, the identity of the JSRV target cell in the lung has not yet been determined. Mature OPA tumors express surfactant protein-C (SP-C) or Clara cell-specific protein (CCSP), which are specific markers of type II pneumocytes or Clara cells, respectively. However, it is unclear whether these are the cell types initially infected and transformed by JSRV or whether the virus targets stem cells in the lung that subsequently acquire a differentiated phenotype during tumor growth. To examine this question, JSRV-infected lung tissue from experimentally infected lambs was studied at early time points after infection. Single JSRV-infected cells were detectable 10 days postinfection in bronchiolar and alveolar regions. These infected cells were labeled with anti-SP-C or anti-CCSP antibodies, indicating that differentiated epithelial cells are early targets for JSRV infection in the ovine lung. In addition, undifferentiated cells that expressed neither SP-C nor CCSP were also found to express the JSRV Env protein. These results enhance the understanding of OPA pathogenesis and may have comparative relevance to human lung cancer, for which samples representing early stages of tumor growth are difficult to obtain.     

Putative phosphatidylinositol 3-kinase (PI3K) binding motifs in ovine betaretrovirus Env proteins are not essential for rodent fibroblast transformation and PI3K/Akt activation

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are simple betaretroviruses that cause epithelial cell tumors in the lower and upper airways of sheep and goats. The envelope (Env) glycoproteins of both viruses can transform rodent and chicken fibroblasts, indicating that they play an essential role in oncogenesis. Previous studies found that a YXXM motif in the Env cytoplasmic tail, a putative docking site for phosphatidylinositol 3-kinase (PI3K) after tyrosine phosphorylation, was necessary for rodent cell transformation but was not required for transformation of DF-1 chicken fibroblasts. Here we show that JSRV and ENTV Env proteins with tyrosine or methionine mutations in the YXXM motif can still transform rodent fibroblasts, albeit with reduced efficiency. Akt was activated in cells transformed by JSRV or ENTV Env proteins and in cells transformed by the proteins with tyrosine mutations. Furthermore, the PI3K-specific inhibitor LY294002 could inhibit Akt activation and cell transformation in all cases, indicating that Akt activation and transformation is PI3K dependent. However, we could not detect tyrosine phosphorylation of JSRV or ENTV Env proteins or an interaction between the Env proteins and PI3K in the transformed cells. We found no evidence for mitogen-activated protein kinase activation in cells that were transformed by the JSRV or ENTV Env proteins. We conclude that ovine betaretrovirus Env proteins transform the rodent fibroblasts by indirectly activating the PI3K/Akt pathway.     

A phosphatidylinositol 3-kinase docking site in the cytoplasmic tail of the Jaagsiekte sheep retrovirus transmembrane protein is essential for envelope-induced transformation of NIH 3T3 cells

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer of sheep known as ovine pulmonary carcinoma. Recently, we have found that the expression of the JSRV envelope (Env) is sufficient to transform mouse NIH 3T3 cells in classical transformation assays. To further investigate the mechanisms of JSRV oncogenesis, we generated a series of envelope chimeras between JSRV and the JSRV-related endogenous retroviruses of sheep (enJSRVs) and assessed them in transformation assays. Chimeras containing the exogenous JSRV SU region and the enJSRV TM region were unable to transform NIH 3T3 cells. Additional chimeras containing only the carboxy-terminal portion of TM (a region that we previously identified as VR3) of the endogenous envelope with SU and the remaining portion of TM from the exogenous JSRV were also unable to transform NIH 3T3 cells. The VR3 region includes the putative membrane-spanning region and cytoplasmic tail of the JSRV TM glycoprotein; this suggested that the cytoplasmic tail of the JSRV Env mediates transformation, possibly via a cell signaling mechanism. Mutations Y590 and M593 in the cytoplasmic tail of the JSRV envelope were sufficient to inhibit the transforming abilities of these constructs. Y590 and M593 are part of a Y-X-X-M motif that is recognized by the phosphatidylinositol 3-kinase (PI-3K). PI-3K initiates a cell signaling pathway that inhibits apoptosis and is required for a number of mitogens during the G(1)-to-S-phase transition of the cell cycle. PI-3K activates Akt by phosphorylation of threonine 308 and serine 473. We detected by Western blot analysis phosphorylated Akt in serum-starved MP1 cells (NIH 3T3 cells transformed by JSRV) but not in the parental NIH 3T3 cells. These data indicate that the cytoplasmic tail of the JSRV TM is necessary for cell transformation and suggest a new mechanism of retroviral transformation. In addition, the ability to dissociate the function of the JSRV envelope to mediate viral entry from its transforming capacity has direct relevance for the design of JSRV-based vectors that target the differentiated epithelial cells of the lungs.     

Lung adenocarcinoma originates from retrovirus infection of proliferating type 2 pneumocytes during pulmonary post-natal development or tissue repair

Jaagsiekte sheep retrovirus (JSRV) is a unique oncogenic virus with distinctive biological properties. JSRV is the only virus causing a naturally occurring lung cancer (ovine pulmonary adenocarcinoma, OPA) and possessing a major structural protein that functions as a dominant oncoprotein. Lung cancer is the major cause of death among cancer patients. OPA can be an extremely useful animal model in order to identify the cells originating lung adenocarcinoma and to study the early events of pulmonary carcinogenesis. In this study, we demonstrated that lung adenocarcinoma in sheep originates from infection and transformation of proliferating type 2 pneumocytes (termed here lung alveolar proliferating cells, LAPCs). We excluded that OPA originates from a bronchioalveolar stem cell, or from mature post-mitotic type 2 pneumocytes or from either proliferating or non-proliferating Clara cells. We show that young animals possess abundant LAPCs and are highly susceptible to JSRV infection and transformation. On the contrary, healthy adult sheep, which are normally resistant to experimental OPA induction, exhibit a relatively low number of LAPCs and are resistant to JSRV infection of the respiratory epithelium. Importantly, induction of lung injury increased dramatically the number of LAPCs in adult sheep and rendered these animals fully susceptible to JSRV infection and transformation. Furthermore, we show that JSRV preferentially infects actively dividing cell in vitro. Overall, our study provides unique insights into pulmonary biology and carcinogenesis and suggests that JSRV and its host have reached an evolutionary equilibrium in which productive infection (and transformation) can occur only in cells that are scarce for most of the lifespan of the sheep. Our data also indicate that, at least in this model, inflammation can predispose to retroviral infection and cancer.     

Transformation of mouse fibroblasts by Jaagsiekte sheep retrovirus envelope does not require phosphatidylinositol 3-kinase

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma, a transmissible lung cancer of sheep. The envelope of JSRV may have oncogenic properties, since it can morphologically transform mouse NIH 3T3 cells and other fibroblast lines. Recently, we found that the cytoplasmic tail of the envelope transmembrane (TM) protein is necessary for transformation, and in particular a consensus binding motif (YXXM) for phosphatidylinositol 3-kinase (PI3K) is important. Moreover, JSRV-transformed cells show phosphorylation (activation) of Akt/protein kinase B, a downstream target of PI3K. In these studies, we directly tested for the involvement of PI3K in transformation by JSRV. Contrary to expectations, four different experiments indicated that PI3K is not necessary for JSRV-induced transformation: (i) cotransfection with a dominant negative truncated form of the PI3K regulatory subunit (Deltap85) did not affect transformation frequency, (ii) cells stably expressing Deltap85 showed the same frequencies of transformation as parental NIH 3T3 cells, (iii) fibroblasts established from double-knockout mice lacking PI3K p85alpha and p85beta could be transformed with JSRV envelope, and (iv) incubation of cells with the PI3K inhibitor LY294002 did not specifically inhibit transformation, nor did the drug reverse transformation of JSRV-transformed cells. One alternate explanation for the lack of transformation by YXXM mutants could be that they were defective in intracellular trafficking. However, confocal microscopy of epitope-tagged envelope proteins of both wild-type and nontransforming YXXM mutants showed a cell surface or plasma membrane localization. While PI3K is not required for JSRV-induced transformation of NIH 3T3 cells, the downstream target Akt kinase was found to be activated (phosphorylated) in JSRV-transformed PI3K-negative cells. Therefore, JSRV envelope can induce PI3K-independent phosphorylation of Akt.     

Non-ulcerative cutaneous lesion in immunodeficient mice with Leishmania amazonensis infection

Cutaneous leishmaniasis begins as papules or nodules at the site of promastigote inoculation. The next key pathogenic event in this disease is the formation of an ulcer at this site. Leishmania infection in immunodeficient mice, however, showed non-ulcerative cutaneous lesions suggesting the involvement of the immune system in ulcer formation. Severe combined immunodeficient (SCID), recombination-activating gene 2 knockout (RAG-2-/-), and immunocompetent mice were inoculated subcutaneously with cultured L. amazonensis promastigotes. Macroscopic nodules appeared at the inoculation site within 2 weeks of infection in all the mice and gradually extended to the surrounding skin tissue. Although nodules of immunocompetent mice ulcerated within 6 weeks, immunodeficient mice did not form ulcers even after 25 weeks of inoculation. These results strongly suggest the importance of functional T and B cells in ulcer formation of cutaneous leishmaniasis and are consistent with clinical features of non-ulcerative cutaneous leishmaniasis in some AIDS patients. The present study also indicates that the L. amazonensis-infected immunodeficient mouse model might be suitable for studying the mechanisms of ulcer formation in cutaneous leishmaniasis.     

小鼠肺腺癌模型的建立及肿瘤病理分析

目的用乙基亚硝脲（ENU）在BABL/c小鼠建立肺腺癌模型并对ENU所诱发的肺腺癌进行病理观察。方法妊娠17d的SPF级母鼠腹腔接受ENU或缓冲液注射,在子代鼠的鼠龄满32周时收获其全肺标本,对肺组织进行常规石蜡半连续切片,HE染色,镜下观察肿瘤病理。结果 ENU经胎盘一次性诱发子代鼠多发性肺肿瘤形成,病理显示这些肿瘤为处于不同发展阶段的腺瘤和腺癌,腺癌的类型有细支气管肺泡癌样腺癌（雌性：5/6,雄性：4/6）和分化不等的腺癌（雌性：4/6,雄性：5/6）,诱癌频率在雌、雄性小鼠均为5/6,癌变频率在雌性16/43,雄性12/31。结论成功建立了小鼠肺腺癌模型,肿瘤病理的多样性提示癌变机制在分子水平的复杂性。     

Retrovirus vectors bearing jaagsiekte sheep retrovirus Env transduce human cells by using a new receptor localized to chromosome 3p21.3

Jaagsiekte sheep retrovirus (JSRV) is a type D retrovirus associated with a contagious lung tumor of sheep, ovine pulmonary carcinoma. Other than sheep, JSRV is known to infect goats, but there is no evidence of human infection. Until now it has not been possible to study the host range for JSRV because of the inability to grow this virus in culture. Here we show that the JSRV envelope protein (Env) can be used to pseudotype Moloney murine leukemia virus (MoMLV)-based retrovirus vectors and that such vectors can transduce human cells in culture. We constructed hybrid retrovirus packaging cells that express the JSRV Env and the MoMLV Gag-Pol proteins and can produce JSRV-pseudotype vectors at titers of up to 10(6) alkaline phosphatase-positive focus-forming units/ml. Using this high-titer virus, we have studied the host range for JSRV, which includes sheep, human, monkey, bovine, dog, and rabbit cells but not mouse, rat, or hamster cells. Considering the inability of the JSRV-pseudotype vector to transduce hamster cells, we used the hamster cell line-based Stanford G3 panel of whole human genome radiation hybrids to phenotypically map the JSRV receptor (JVR) gene within the p21.3 region of human chromosome 3. JVR is likely a new retrovirus receptor, as none of the previously identified retrovirus receptors localizes to the same position. Several chemokine receptors that have been shown to serve as coreceptors for lentivirus infection are clustered in the same region of chromosome 3; however, careful examination shows that the JSRV receptor does not colocalize with any of these genes.