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Multiple Integrated Copies and High-Level Production of the Human Retrovirus XMRV (Xenotropic Murine Leukemia Virus-Related Virus) from 22Rv1 Prostate Carcinoma Cells 
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下载免费PDF全文 Emily C. Knouf Michael J. Metzger Patrick S. Mitchell Jason D. Arroyo John R. Chevillet Muneesh Tewari A. Dusty Miller 《Journal of virology》2009,83(14):7353-7356
The human retrovirus XMRV (xenotropic murine leukemia virus-related virus) is associated with prostate cancer, most frequently in humans with a defect in the antiviral defense protein RNase L, suggesting a role for XMRV in prostate carcinogenesis. However, XMRV has not been found in prostate carcinoma cells. Here we show that 22Rv1 prostate carcinoma cells produce high-titer virus that is nearly identical in properties and sequence to XMRV isolated by others and consist primarily of a single clone of cells with at least 10 integrated copies of XMRV, warranting further study of a possible role for XMRV integration in carcinogenesis.The association of human prostate cancer with mutations that impair the function of the antiviral defense protein RNase L has suggested a role for virus in prostate cancer. Indeed, analysis of cDNA from prostate tumors using a DNA microarray (Virochip) containing conserved DNA sequences from all known virus families indicated the presence of a novel gammaretrovirus in 40% of prostate cancer patients having homozygous R462Q mutations in RNase L (16). Cloning and sequencing of the virus revealed a close similarity to mouse xenotropic retroviruses; therefore, the new virus was named xenotropic murine leukemia virus-related virus (XMRV) (16). Importantly, XMRV has been found integrated into human genomic DNA from tumor-bearing prostatic tissue samples from 11 patients, showing that XMRV can indeed infect humans and is not a laboratory contaminant (2, 7). However, the possibility that XMRV plays a role in prostate cancer is weakened by the lack of an obvious oncogene in XMRV and by the finding that XMRV was not associated with prostate carcinoma cells but instead with tumor stromal cells, arguing against direct viral oncogenesis or insertional activation of oncogenes in the carcinoma cells by XMRV (16). Here we describe the detection of multiple integrated copies and high-level production of XMRV from 22Rv1 prostate carcinoma cells, which were derived from a primary prostatic carcinoma (14, 15). These cells secrete prostate-specific antigen, express an androgen receptor, and are responsive to dihydroxytestosterone (15), evidence that they are indeed of prostate epithelial cell origin.Electron microscopic analysis of culture medium from 22Rv1 prostate carcinoma cells (ATCC CRL-2505) revealed the presence of gammaretrovirus-like particles (Fig. (Fig.1).1). To detect and characterize the biological activity of the presumptive virus, we used a marker rescue assay (10). In brief, HTX cells (an approximately diploid subclone of human HT-1080 fibrosarcoma cells) transduced with the retroviral vector LAPSN (HTX/LAPSN cells) were exposed to culture medium conditioned by confluent layers of 22Rv1 cells for 24 h. The HTX/LAPSN cells were passaged for 2 weeks to allow virus spread. Next the cells were assayed for production of the LAPSN vector by measuring transfer of the alkaline phosphatase (AP) gene carried by the LAPSN vector to naïve HTX target cells and to Mus dunni tail fibroblast cells. The Mus dunni cells are wild mouse cells that are infectible by many gammaretroviruses, including xenotropic retroviruses (12). Medium samples from 22Rv1 cells originally obtained from the ATCC and maintained in the M. Tewari lab and from 22Rv1 cells freshly obtained from the ATCC tested highly positive for the presence of replication-competent virus in this marker rescue assay using either HTX or Mus dunni cells as targets for infection (data not shown).Open in a separate windowFIG. 1.Presence of retrovirus-like particles in culture medium from 22Rv1 cells. Medium was harvested from 22Rv1 cells, clarified by centrifugation at 16,500 × g for 20 min, filtered through 0.22-μm-pore-size filters, and centrifuged at 120,000 × g for 70 min, and the pelleted material was analyzed by transmission electron microscopy. Arrows indicate retrovirus-like particles with dark cores surrounded by a membrane.To provide a quantitative measure of the virus released by the 22Rv1 cells, we used an S+L− helper virus assay (11). This assay measures the ability of the test virus to rescue a transforming virus from PG-4 cat cells and induce foci of transformation. Assay of medium conditioned for 24 h by 22Rv1 cells maintained in the Tewari lab and by 22Rv1 cells freshly obtained from the ATCC gave titers from 2 × 106 to 107 focus-forming units (FFU) per ml, revealing high-titer virus production by 22Rv1 cells and showing that the 22Rv1 virus can infect and replicate in cat cells.XMRV is unable to infect Chinese hamster ovary (CHO) cells, but expression of the xenotropic and polytropic retrovirus receptor Xpr1 from human cells (1) in CHO cells renders them susceptible to XMRV infection (2). Xenotropic viruses from mice were named on the basis of their ability to infect “foreign” cells but not those of laboratory mice; however, the ability of XMRV to infect laboratory mouse cells has not been determined. To test whether the virus released from 22Rv1 cells has these properties of mouse xenotropic viruses, we used virus generated in the marker rescue assay to infect hamster and mouse cells that expressed or did not express human Xpr1 (Table (Table1).1). For a control, we measured infection by the LAPSN vector pseudotyped with the NZB mouse xenotropic retrovirus [LAPSN(NZB)] produced from Mus dunni/LAPSN+NZB cells (12). We found that infection of the mouse and hamster cells by the 22Rv1 virus-pseudotyped LAPSN vector required the presence of Xpr1. Infection of mouse cells by LAPSN(NZB) virus also required the presence of Xpr1, but infection of hamster cells by LAPSN(NZB) was facilitated by but did not require Xpr1. These results show that the phenotype of the 22Rv1 virus is identical to that previously reported for XMRV (2) but is somewhat different from the typical mouse xenotropic retrovirus NZB. In addition, our results show that XMRV can infect human, cat, and wild mouse cells, but it does not infect laboratory mouse or Chinese hamster cells.
Open in a separate windowaConditioned medium was harvested from confluent layers of 22Rv1 cells. Medium samples were filtered through 0.45-μm-pore-size filters and added to HTX/LAPSN cells, and the cells were passaged for 2 weeks to allow virus spread. Medium was harvested and filtered 12 to 24 h after feeding confluent layers of HTX/LAPSN cells exposed to medium from the 22Rv1 cells, from naïve HTX/LAPSN cells (no helper virus), and from Mus dunni/LAPSN cells infected with the NZB mouse xenotropic retrovirus.bTarget cells were exposed to various amounts of the harvested medium and were stained for alkaline phosphatase-positive (AP+) cell foci 2 days later. Target cells included NIH 3T3 mouse cells, NIH 3T3 cells transduced with a retrovirus vector expressing the human xenotropic retrovirus receptor Xpr1 (LhXprSN) (1), CHO cells transduced with a control retrovirus vector LXSN, and CHO cells transduced with the retrovirus vector expressing Xpr1. LAPSN vector titer is expressed as AP+ FFU per ml of medium.c22Rv1 cells obtained from the ATCC and maintained in the M. Tewari lab (MT lab) or from 22Rv1 cells freshly obtained from the ATCC (ATCC).The 22Rv1 cells were derived from cells that had been grown by xenotransplantation in nude mice (13, 14, 17); therefore, it was possible that the 22Rv1 virus was acquired from mice. To further establish the identity of the 22Rv1 virus, we cloned and sequenced a 600-bp region of the 22Rv1 virus gag gene that is different in XMRV and related mouse retroviruses by using previously described GAG-OF and GAG-OR primers (16) to PCR amplify the region from reverse-transcribed viral RNA. A BLAST search of the nonredundant GenBank sequences for sequences similar to two cloned sequences (EK1 and EK2 [GenBank accession numbers FJ907198 and FJ907199, respectively]) revealed nearly exact matches to XMRV clone VP42, and phylogenetic analysis revealed that the 22Rv1 sequences clearly cluster with all previously cloned XMRV sequences (Fig. (Fig.2).2). From these data, we conclude that the 22Rv1 virus is XMRV and is not a mouse xenotropic virus acquired during passage of the cells in culture or in mice (see additional supporting data in the Addendum in Proof).Open in a separate windowFIG. 2.Phylogenetic tree of 22Rv1 gag sequences (clones EK1 and EK2), most closely related GenBank sequences, and one more distant sequence (polytropic mouse endogenous retrovirus [GenBank accession number FJ544576.1]). Sequences were aligned using ClustalX, and an unrooted neighbor-joining tree was generated using this alignment and MEGA version 4 software. Bootstrap values (500 replicates) are shown as percentages, and the scale bar indicates evolutionary distance in base substitutions per site. mERV, mouse endogenous retrovirus; MRV, mouse AIDS-related virus; chr.12, chromosome 12.We were interested to see whether other commonly used prostate carcinoma cell lines produce XMRV. Previous work indicates that LNCaP and DU145 prostate carcinoma cells do not produce XMRV (2). For our analysis, medium exposed to test cells for 24 h was assayed for the presence of replication-competent retrovirus using the S+L− assay described above. LNCaP (5) and PC-3 (6) cells tested negative for virus production (<1 FFU/ml), while VCaP cells (8) tested weakly positive for virus production (20 FFU/ml), and the transformed foci formed slowly, indicating poor replication of this virus in feline S+L− cells. In contrast, XMRV virus from 22Rv1 cells gave titers of 2 × 106 to 107 FFU/ml, and the foci formed rapidly, indicating efficient replication of XMRV in the feline cells.We further characterized the virus from VCaP cells by marker rescue assay as follows. HTX/LAPSN and Mus dunni/LAPSN cells were exposed to filtered medium from VCaP cells and passaged for 2 weeks to allow virus spread, and medium from these cells was tested for the presence of the LAPSN vector by assay on naïve HTX and Mus dunni cells. High-level LAPSN vector production was detected from the VCaP medium-exposed Mus dunni/LAPSN cells when measured on Mus dunni target cells (5 × 106 AP+ FFU/ml), but when measured on HTX cells, the titer was 1,000-fold lower (6 × 103 AP+ FFU/ml). No LAPSN vector production was detected from the VCaP medium-exposed HTX/LAPSN cells when measured on Mus dunni or on HTX cells (<1 AP+ FFU/ml). These results show that the VCaP virus replicates well in Mus dunni cells but only poorly infects and replicates in HTX cells, unlike XMRV, which efficiently infects and replicates in HTX cells (Table (Table11 and data not shown). Thus, of the five prostate carcinoma cells tested, only the 22Rv1 cells produce XMRV.XMRV is most frequently detected in humans with homozygous R462Q mutations in RNase L (16). To determine whether this mutation was present in 22Rv1 cells, we used PCR to amplify this region of the RNASEL gene from 22Rv1 cell DNA, HTX cell DNA, and from a 1:1 mixture of the two DNAs. Sequencing of the resulting products revealed only the wild-type amino acid codon (CGA) in HTX cell DNA, only the R462Q mutant codon (CAA) in 22Rv1 cell DNA, and a mixture of the two codons in the mixed DNA sample, which shows that simultaneous detection of both codons was possible. These results show that 22Rv1 cells are homozygous for the R462Q mutant RNASEL allele.Oncogenesis by retroviruses can result from insertional activation of oncogenes followed by outgrowth of clonal tumor cell lines that are often marked by multiple retrovirus integrations. To examine XMRV integration sites in the 22Rv1 cells, we performed Southern analysis of 22Rv1 cell DNA by using our cloned XMRV gag sequences as a probe and by using restriction enzymes that cut on both sides of the XMRV gag region (KpnI) or that cut on one side of the gag region and at variable sites in surrounding human genomic DNA (BamHI) (Fig. (Fig.3).3). KpnI digestion produced the expected 2.81-kb band (Fig. (Fig.3),3), and phosphorimager analysis indicated a band intensity corresponding to at least 10 integrated virus copies per cell. BamHI digestion revealed at least 10 bands at approximately single-copy-per-cell levels, indicating that the 22Rv1 cell population consists primarily of a clone of cells with at least 10 XMRV integration sites. Note that BamHI also creates an internal 2.13-kb viral fragment that hybridizes with the gag probe (Fig. (Fig.3).3). This fragment contains only 10% of the gag probe sequence, and therefore, the observed band is not as intense as the KpnI band but is clearly more intense than the other bands in this lane. We found no hybridization of the gag probe to DNA from human HTX cells, consistent with the fact that no endogenous retroviruses closely related to XMRV are present in the human genome. In summary, these results are consistent with the restriction enzyme patterns predicted by previous sequencing of XMRV and are consistent with but do not prove a model of prostate carcinogenesis involving insertional activation of cellular oncogenes by XMRV.Open in a separate windowFIG. 3.Southern analysis of genomic DNA from 22Rv1 and HTX cells using an XMRV gag probe. Both panels shown are from the same exposure of the same blot with some internal lanes removed. At the bottom of the figure is a scale diagram of XMRV based on the sequence of the VP42 XMRV clone (GenBank accession number DQ241302) with sizes indicated for the internal restriction fragments that hybridize with the gag probe (2.81 and 2.13 kb). Bands in the blot corresponding to these fragments are marked with asterisks. Copy number standards were made using a plasmid containing the gag probe sequences cut with a restriction enzyme that cuts once in the bacterial plasmid sequences and using an estimate of 6 × 109 bp for the genome size of 22Rv1 cells. LTR, long terminal repeat.In summary, we report here the first identification of XMRV in prostate carcinoma cells. Earlier studies found the virus in prostate tumor stromal cells by fluorescence in situ hybridization and by immunohistochemistry using Gag-specific antibodies (2) or in bulk tumor material by PCR without determination of the infected cell type (4). Unfortunately, we are unable to determine the extent of carcinoma cell infection in the original tumor, and it is possible that the 22Rv1 carcinoma cells were infected by virus produced by tumor stromal cells during passage as a xenograft in mice. Interestingly, we found that the 22Rv1 carcinoma cell line is primarily composed of a single clonal cell line marked by ≥10 XMRV integration sites, consistent with a model of carcinogenesis involving insertional activation of oncogenes by XMRV. However, this result is also consistent with the presence of multiple integrations in a particular cell followed by outgrowth of the cell because of oncogenic events unrelated to the virus integrations, or more trivially, to clonal outgrowth during passage of the cells in mice or in culture, although we never cloned the 22Rv1 cells and the literature indicates these and the parental cells have always been grown as a bulk population (13-15, 17). Initial studies by others have found no common XMRV integration sites within or near proto-oncogenes or tumor suppressor genes in tumor tissue from nine prostate cancer patients (7), arguing against a role for virus insertion in prostate cancer. Accordingly, it is possible that XMRV has other roles in prostate cancer, such as the alteration of stromal cells to facilitate cancer development or the generation of aneuploid genetically unstable cells by virus-mediated cell fusion (3).Finally, production of XMRV by 22Rv1 and potentially other prostate cancer cell lines should be carefully considered from the standpoint of possible virus transmission to laboratory personnel, to other cells cultured in parallel, and as a confounding factor in the interpretation of experimental results. For example, 22Rv1 cells have been reported to produce 80- to 150-nm “exosomes” (9) which look very much like the presumed ∼100-nm retrovirus particles shown in Fig. Fig.1,1, and the possible role of XMRV in the phenomena ascribed to exosomes will need clarification. 相似文献
TABLE 1.
22Rv1 retrovirus host range and receptor usea| Helper virus | LAPSN vector titer (AP+ FFU/ml) on target cellsb:
| |||
|---|---|---|---|---|
| NIH 3T3 | NIH 3T3/Xpr1 | CHO | CHO/Xpr1 | |
| 22Rv1 (MT lab)c | <1 | 100 | <1 | 3 × 105 |
| 22Rv1 (ATCC)c | <1 | 100 | 2 | 3 × 105 |
| NZB xenotropic | <5 | 100 | 3 × 103 | 6 × 105 |
| None | <1 | <1 | <1 | <1 |
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魏志涛杨勇 《现代生物医学进展》2012,12(25):4998-5000
异嗜性小鼠白血病病毒相关病毒(XMRV)是第一个能够感染人的γ型逆转录病毒。由于逆转录病毒具有插入宿主染色体引发基因突变的特性,以及部分γ型逆转录病毒携带原癌基因并能诱发癌症,致使XMRV感染与人类癌症发病之间的关系受到广泛关注。西方研究表明XMRV感染与前列腺癌和慢性疲劳综合症的发病有一定的相关性,因此成为研究热点。但各地报告的检测结果却不尽相同,甚至有矛盾之处。XMRV致瘤性可能是能够激活内源性原癌基因,从而导致细胞转化。在我国,目前尚未见XMRV感染与人类疾病关系的相关研究报道。XMRV感染的地区间或人群间差异性也有待研究。其感染的途径及临床可普及应用的检测手段也未被阐明,在我国不同地区不同类型人群中的感染率、与疾病的关系、对我国人口健康的潜在影响,均有待研究。 相似文献
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Sushma Bhosle Suganthi Suppiah Ross Molinaro Yuying Liang Rebecca Arnold William Diehl Natalia Makarova Jerry Blackwell John Petros Dennis Liotta Eric Hunter Hinh Ly 《Journal of virology》2010,84(13):6288-6296
The newly identified retrovirus—the xenotropic murine leukemia virus-related virus (XMRV)—has recently been shown to be strongly associated with familial prostate cancer in humans (A. Urisman et al., PLoS Pathog. 2:e25, 2006). While that study showed evidence of XMRV infection exclusively in the prostatic stromal fibroblasts, a recent study found XMRV protein antigens mainly in malignant prostate epithelial cells (R. Schlaberg et al., Proc. Natl. Acad. Sci. U. S. A. 106:16351-16356, 2009). To help elucidate the mechanisms behind XMRV infection, we show that prostatic fibroblast cells express Xpr1, a known receptor of XMRV, but its expression is absent in other cell lines of the prostate (i.e., epithelial and stromal smooth muscle cells). We also show that certain amino acid residues located within the predicted extracellular loop (ECL3 and ECL4) sequences of Xpr1 are required for efficient XMRV entry. Although we found strong evidence to support XMRV infection of prostatic fibroblast cell lines via Xpr1, we learned that XMRV was indeed capable of infecting cells that did not necessarily express Xpr1, such as those of the prostatic epithelial and smooth muscle origins. Further studies suggest that the expression of Xpr1 and certain genotypes of the RNASEL gene, which could restrict XMRV infection, may play important roles in defining XMRV tropisms in certain cell types. Collectively, our data reveal important cellular determinants required for XMRV entry into different human prostate cells in vitro, which may provide important insights into the possible role of XMRV as an etiologic agent in human prostate cancer.Prostate cancer is the most common male malignancy in Western countries and the second most common cause of cancer-related deaths in males worldwide (15, 24). The known risk factors for prostate cancer are hormones (i.e., androgens), diet, sex, and race, as well as environmental and genetic factors (27). A recent study suggests that susceptibility to prostate cancer can be influenced by the genetic variations associated with an antagonistic coevolution, which occurs between a specific host locus (RNASEL), known to be involved in antiviral innate immune defense, and a viral pathogen (38). Indeed, several epidemiologic studies have supported the involvement of the RNASEL gene in the prostate cancer etiology (4, 5, 30, 31), whereas other studies do not (9, 22, 34, 43). Some studies have reported that individuals with a single mutated copy of the RNASEL gene have a 50% increased risk for prostate cancer, whereas those with homozygous mutant RNASEL alleles have a 2-fold-increased risk of prostate cancer (5).The RNASEL gene encodes for the RNase L protein, a constitutively expressed latent endoribonuclease, which mediates the interferon-inducible 2-5A system against viral and/or cellular double-stranded RNAs (8, 16, 20, 23, 49, 50). The RNase L “Q” variant allele (R462Q) shows a 3-fold decrease in catalytic activity compared to the wild-type enzyme (5, 44). The possible association of mutant RNASEL alleles with human prostate cancers suggests an enhanced susceptibility of prostate tissues to a viral agent. This hypothesis has led to the recent identification of a new human retrovirus, xenotropic murine leukemia virus (MuLV)-related virus (XMRV), in 40% of prostate cancer patients with the QQ variant alleles of RNASEL compared to 1.5% among heterozygous (RQ) and wild-type (RR) RNASEL carriers (41). XMRV virus infection appears to be susceptible to inhibition by interferon and its downstream effector RNase L protein (7). However, a recent study has provided some evidence to show that XMRV infection is independent of the RNASEL genotype (34), suggesting that population differences and/or other environmental or genetic factors may influence the impact of RNASEL on prostate cancer development.The XMRV genome is 8,185 nucleotides in length and shares up to 95% overall nucleotide sequence identity with known xenotropic MuLVs (41). One receptor for xenotropic MuLVs is Xpr1, a 696-amino-acid protein with multiple transmembrane-spanning domains (2). Expression of this protein in Chinese hamster ovary (CHO) cells that are not known to express Xpr1 endogenously confers an enhanced susceptibility of these cells to xenotropic MuLV infection (2). Infection of hamster and mouse cells with XMRV-like virus that is derived from a prostate cancer cell line (22Rv1) also requires Xpr1 as a receptor (18). Earlier studies have demonstrated the importance of certain residues located within the putative third and fourth extracellular loops (ECL3 and ECL4) of Mus dunni''s Xpr1 in conferring infection by xenotropic MuLVs (25). Furthermore, it has been shown that the specific and common receptor determinants for xenotropic and polytropic murine retroviruses are simultaneously present in discrete domains of a single Xpr1 protein (42). In the present study, we characterized for the first time the important molecular determinants on Xpr1 required for XMRV infection and investigated the role of RNase L in restricting XMRV infection of various human prostate cancer and noncancerous cell lines. 相似文献
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Sanggu Kim Alice Rusmevichientong Beihua Dong Roland Remenyi Robert H. Silverman Samson A. Chow 《PloS one》2010,5(4)
Xenotropic murine leukemia virus (MLV)-related virus (XMRV) is a new human retrovirus associated with prostate cancer and chronic fatigue syndrome. The causal relationship of XMRV infection to human disease and the mechanism of pathogenicity have not been established. During retrovirus replication, integration of the cDNA copy of the viral RNA genome into the host cell chromosome is an essential step and involves coordinated joining of the two ends of the linear viral DNA into staggered sites on target DNA. Correct integration produces proviruses that are flanked by a short direct repeat, which varies from 4 to 6 bp among the retroviruses but is invariant for each particular retrovirus. Uncoordinated joining of the two viral DNA ends into target DNA can cause insertions, deletions, or other genomic alterations at the integration site. To determine the fidelity of XMRV integration, cells infected with XMRV were clonally expanded and DNA sequences at the viral-host DNA junctions were determined and analyzed. We found that a majority of the provirus ends were correctly processed and flanked by a 4-bp direct repeat of host DNA. A weak consensus sequence was also detected at the XMRV integration sites. We conclude that integration of XMRV DNA involves a coordinated joining of two viral DNA ends that are spaced 4 bp apart on the target DNA and proceeds with high fidelity. 相似文献
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David Palesch Mohammad Khalid Christina M. Stürzel Jan Münch 《Applied and environmental microbiology》2014,80(8):2617-2622
Xenotropic murine leukemia virus-related virus (XMRV) represents a novel γ-retrovirus that is capable of infecting human cells and has been classified as a biosafety level 2 (BSL-2) organism. Hence, XMRV represents a potential risk for personnel in laboratories worldwide. Here, we measured the stability of XMRV and its susceptibility to alcohol-based disinfectants. To this end, we exposed an infectious XMRV reporter virus encoding a secretable luciferase to different temperatures, pH values, and disinfectants and infected XMRV-permissive Raji B cells to measure residual viral infectivity. We found that 1 min treatment of XMRV particles at 60°C is sufficient to reduce infectivity by 99.9%. XMRV infectivity was maximal at a neutral pH but was reduced by 86% at pH 4 and 99.9% at pH 10. The common hand and surface disinfectants ethanol and isopropanol as well as the cell fixation reagent paraformaldehyde abrogated XMRV infectivity entirely, as indicated by a reduction of infectivity exceeding 99.99%. Our findings provide evidence of specific means to inactivate XMRV. Their application will help to prevent unintended XMRV contamination of cell cultures in laboratories and minimize the risk for laboratory personnel and health care workers to become infected with this biosafety level 2 organism. 相似文献
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Tsai-Der Chuang Siu-Ju Chen Fen-Fen Lin Suresh Veeramani Satyendra Kumar Surinder K. Batra Yaping Tu Ming-Fong Lin 《The Journal of biological chemistry》2010,285(31):23598-23606
Cellular prostatic acid phosphatase (cPAcP), an authentic tyrosine phosphatase, is proposed to function as a negative growth regulator of prostate cancer (PCa) cells in part through its dephosphorylation of ErbB-2. Nevertheless, the direct interaction between cPAcP and ErbB-2 has not been shown nor the specific dephosphorylation site of ErbB-2 by cPAcP. In this report, our data show that the phosphorylation level of ErbB-2 primarily at Tyr1221/2 correlates with the growth rate of both LNCaP and MDA PCa2b human PCa cells. Further, cPAcP reciprocally co-immunoprecipitated with ErbB-2 in a non-permissive growth condition. Expression of wild type cPAcP, but not inactive mutant, by cDNA in cPAcP-null LNCaP C-81 cells results in decreased tyrosine phosphorylation of ErbB-2 including Tyr1221/2. Concurrently, Tyr317 phosphorylation of p52Shc, proliferating cell nuclear antigen expression, and cell growth are decreased in these cells. Conversely, decreased cPAcP expression by short hairpin RNA in LNCaP C-33 cells was associated with elevated phosphorylation of ErbB-2 initially at Tyr1221/2. Its downstream p52Shc, ERK1/2, Akt, Src, STAT-3, and STAT-5 were activated, and cell proliferation, proliferating cell nuclear antigen, and cyclin D1 expression were increased. Stable subclones of C-33 cells by small interfering PAcP had elevated Tyr1221/2 phosphorylation of ErbB-2 and exhibited androgen-independent growth and increased tumorigenicity in xenograft female animals. In summary, our data together indicate that in prostate epithelia, cPAcP interacts with and dephosphorylates ErbB-2 primarily at Tyr1221/2 and hence blocks downstream signaling, leading to reduced cell growth. In PCa cells, decreased cPAcP expression is associated with androgen-independent cell proliferation and tumorigenicity as seen in advanced hormone-refractory prostate carcinomas. 相似文献
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Prostatic acid phosphatase (PAP) expression increases proportionally with prostate cancer progression, making it useful in prognosticating intermediate to high-risk prostate cancers. A novel ligand that can specifically bind to PAP would be very helpful for guiding prostate cancer therapy. RNA aptamers bind to target molecules with high specificity and have key advantages such as low immunogenicity and easy synthesis. Here, human PAP-specific aptamers were screened from a 2′-fluoropyrimidine (FY)-modified RNA library by SELEX. The candidate aptamer families were identified within six rounds followed by analysis of their sequences and PAP-specific binding. A gel shift assay was used to identify PAP binding aptamers and the 6N aptamer specifically bound to PAP with a Kd value of 118 nM. RT-PCR and fluorescence labeling analyses revealed that the 6N aptamer bound to PAP-positive mammalian cells, such as PC-3 and LNCaP. IMR-90 negative control cells did not bind the 6N aptamer. Systematic minimization analyses revealed that 50 nucleotide sequences and their two hairpin structures in the 6N 2′-FY RNA aptamer were equally important for PAP binding. Renewed interest in PAP combined with the versatility of RNA aptamers, including conjugation of anti-cancer drugs and nano-imaging probes, could open up a new route for early theragnosis of prostate cancer. 相似文献
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Analysis of the Disease Potential of a Recombinant Retrovirus Containing Friend Murine Leukemia Virus Sequences and a Unique Long Terminal Repeat from Feline Leukemia Virus 下载免费PDF全文
下载免费PDF全文 Kazuo Nishigaki Charlotte Hanson Delores Thompson Takashi Yugawa Masaharu Hisasue Hajime Tsujimoto Sandra Ruscetti 《Journal of virology》2002,76(3):1527-1532
We have molecularly cloned a feline leukemia virus (FeLV) (clone 33) from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV proviruses from other cats with AML, contains an unusual structure in its U3 region upstream of the enhancer (URE) consisting of three tandem direct repeats of 47 bp. To test the disease potential and specificity of this unique FeLV LTR, we replaced the U3 region of the LTR of the erythroleukemia-inducing Friend murine leukemia virus (F-MuLV) with that of FeLV clone 33. When the resulting virus, F33V, was injected into newborn mice, almost all of the mice eventually developed hematopoietic malignancies, with a significant percentage being in the myeloid lineage. This is in contrast to mice injected with an F-MuLV recombinant containing the U3 region of another FeLV that lacks repetitive URE sequences, none of which developed myeloid malignancies. Examination of tumor proviruses from F33V-infected mice failed to detect any changes in FeLV U3 sequences other than that in the URE. Like F-MuLV-infected mice, those infected with the F-MuLV/FeLV recombinants were able to generate and replicate mink cell focus-inducing viruses. Our studies are consistent with the idea that the presence of repetitive sequences upstream of the enhancer in the LTR of FeLV may favor the activation of this promoter in myeloid cells and contribute to the development of malignancies in this hematopoietic lineage. 相似文献
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Friend spleen focus-forming virus (F-SFFV) is a replication-defective acutely leukemogenic mouse retrovirus and encodes an envelope protein (Env)-like membrane glycoprotein (gp55) in its defective env gene, which is responsible for the early stage of the viral leukemogenesis. Gp55 is a modified Env protein and contains a polytropic mink cell focus-inducing (MCF) murine leukemia virus (MuLV) Env gp70-derived sequence in its amino-terminal region. To evaluate the possibility that the presumed binding of gp55 to an MCF MuLV receptor protein has some role in leukemogenesis, we examined the biological activities of a mutant gp55 (XE gp55), which has a xenotropic MuLV Env gp70 amino-terminal region. XE gp55 displayed almost the same biological activities as the wild-type gp55, excluding the above possibility. 相似文献
16.
Jenifer H. Anklesaria Dhanashree D. Jagtap Bhakti R. Pathak Kaushiki M. Kadam Shaini Joseph Smita D. Mahale 《PloS one》2013,8(3)
Prostate Secretory Protein of 94 amino acids (PSP94) is one of the major proteins present in the human seminal plasma. Though several functions have been predicted for this protein, its exact role either in sperm function or in prostate pathophysiology has not been clearly defined. Attempts to understand the mechanism of action of PSP94 has led to the search for its probable binding partners. This has resulted in the identification of PSP94 binding proteins in plasma and seminal plasma from human. During the chromatographic separation step of proteins from human seminal plasma by reversed phase HPLC, we had observed that in addition to the main fraction of PSP94, other fractions containing higher molecular weight proteins also showed the presence of detectable amounts of PSP94. This prompted us to hypothesize that PSP94 could be present in the seminal plasma complexed with other protein/s of higher molecular weight. One such fraction containing a major protein of ∼47 kDa, on characterization by mass spectrometric analysis, was identified to be Prostatic Acid Phosphatase (PAP). The ability of PAP present in this fraction to bind to PSP94 was demonstrated by affinity chromatography. Co-immunoprecipitation experiments confirmed the presence of PSP94-PAP complex both in the fraction studied and in the fresh seminal plasma. In silico molecular modeling of the PSP94-PAP complex suggests that β-strands 1 and 6 of PSP94 appear to interact with domain 2 of PAP, while β-strands 7 and 10 with domain 1 of PAP. This is the first report which suggests that PSP94 can bind to PAP and the PAP-bound PSP94 is present in human seminal plasma. 相似文献
17.
Rebeca Kawahara Saulo Recuero Fabio C. S. Nogueira Gilberto B. Domont Katia R. M. Leite Miguel Srougi Morten Thaysen‐Andersen Giuseppe Palmisano 《Proteomics》2019,19(21-22)
The histology‐based Gleason score (GS) of prostate cancer (PCa) tissue biopsy is the most accurate predictor of disease aggressiveness and an important measure to guide treatment strategies and patient management. The variability associated with PCa tumor sampling and the subjective determination of the GS are challenges that limit accurate diagnostication and prognostication. Thus, novel molecular signatures are needed to distinguish between indolent and aggressive forms of PCa for better patient management and outcomes. Herein, label‐free LC‐MS/MS proteomics is used to profile the proteome of 50 PCa tissues spanning five grade groups (n = 10 per group) relative to tissues from individuals with benign prostatic hyperplasia (BPH). Over 2000 proteins are identified albeit at different levels between and within the patient groups, revealing biological processes associated with specific grades. A panel of 11 prostate‐derived proteins including IGKV3D‐20, RNASET2, TACC2, ANXA7, LMOD1, PRCP, GYG1, NDUFV1, H1FX, APOBEC3C, and CTSZ display the potential to stratify patients from low and high PCa grade groups. Parallel reaction monitoring of the same sample cohort validate the differential expression of LMOD1, GYG1, IGKV3D‐20, and RNASET2. The four proteins associated with low and high PCa grades reported here warrant further exploration as candidate biomarkers for PCa aggressiveness. 相似文献
18.
Maria Azrad Kui Zhang Robin T. Vollmer John Madden Thomas J. Polascik Denise C. Snyder Mack T. Ruffin Judd W. Moul Dean Brenner Robert W. Hardy Wendy Demark-Wahnefried 《PloS one》2012,7(12)
Previous observational studies have reported associations between prostate cancer and alpha-linolenic acid (ALA). However, few investigations have been able to study this relationship prospectively and in well-controlled settings. Moreover, no studies have determined whether single nucleotide polymorphisms (SNPs) that influence ALA metabolism are associated with this common cancer. The purpose of this study was to explore associations between prostatic levels of ALA, SNPs and prostate cancer-specific biomarkers in samples collected from a previous randomized clinical trial conducted using a presurgical model and which tested the effects of flaxseed supplementation, a rich source of ALA, prior to prostatectomy (n = 134). Serum prostate-specific antigen (PSA) was determined and immunohistochemistry was used to assess tumor proliferation rate (Ki67). Prostatic ALA was determined with gas chromatography. Seven previously identified SNPs associated with delta-6 desaturase activity (rs99780, rs174537, rs174545, rs174572, rs498793, rs3834458 and rs968567) were tested for associations with prostatic ALA, PSA and Ki67. Despite consuming seven times more ALA per day, men in the flaxseed arm had similar amounts of prostatic ALA relative to men not consuming flaxseed. In unadjusted analysis, there were significant positive associations between prostatic ALA and PSA (ρ = 0.191, p = 0.028) and Ki67 (ρ = 0.186, p = 0.037). After adjusting for covariates (flaxseed, age, race, BMI and statin-use) the association between ALA and PSA remained (p = 0.004) but was slightly attenuated for Ki67 (p = 0.051). We did not observe associations between any of the SNPs studied and prostatic ALA; however, in models for PSA there was a significant interaction between rs498793 and ALA and for Ki67 there were significant interactions with ALA and rs99780 and rs174545. Independent and inverse associations were observed between rs174572 and Ki67. This study provides evidence that prostatic ALA, independent of the amount of ALA consumed, is positively associated with biomarkers of aggressive prostate cancer and that genetic variation may modify this relationship. 相似文献
19.
Cell Surface Antigens Associated with Murine Leukemia Virus: Definition of the GL and GT Antigenic Systems 总被引：4，自引：0，他引：4 下载免费PDF全文
下载免费PDF全文 Two new serological specificities were identified on the surface of murine leukemia virus (MuLV)-infected cells by direct and absorption immunofluorescence tests. Both antigens were detected with antisera prepared in rats that were growing transplants of syngenic MuLV-induced leukemias. Antigen GL was defined with the AKR leukemia K36 as the test cell; antigen GT was defined with the W/Fu leukemia C58(NT)D as the test cell. GL and GT antigens were serologically and genetically independent of the MuLV-induced Gross and GIX cell-surface antigens. GL and GT antigens were found in normal lymphoid cells of mice from high-leukemic strains, but not in lymphoid tissues of mice from most low-leukemic strains. Tumors and leukemias of mice of low-leukemic strains often were GL and GT positive. Similarly, infection of normal cells with MuLV resulted in expression of GL and GT. With ferritin-labeled antibody the GL and GT antigens were observed on virus-free segments of the cell surface. Genetically, GL and GT antigens were each controlled by two dominant unlinked genes in AKR mice; these same antigens were each controlled by three or more dominant unlinked genes in C58 mice. Penetrance of GL and GT regulatory genes was dependent upon the Fv-1 genotype of the host. Expression of GL antigen was closely associated with virus production, whereas expression of GT antigen was less closely associated. 相似文献
20.
《Journal of molecular biology》2023,435(13):168096
Human papilloma virus (HPV) infections are associated with almost all cervical cancers and to a lower extend also with anogenital or oropharyngeal cancers. HPV proteins expressed in HPV-associated tumors are attractive antigens for cancer vaccination strategies as self-tolerance, which is associated with most endogenous tumor-associated antigens, does not need to be overcome. In this study, we generated a live attenuated cancer vaccine based on the chimeric vesicular stomatitis virus VSV-GP, which has previously proven to be a potent vaccine vector and oncolytic virus. Genes at an earlier position in the genome more to the 3′ end are expressed stronger compared to genes located further downstream. By inserting an HPV16-derived antigen cassette consisting of E2, E6 and E7 into VSV-GP either at first (HPVp1) or fifth (HPVp5) position in VSV-GP’s genome we aimed to analyze the effect of vaccine antigen position and consequently expression level on viral fitness, immunogenicity, and anti-tumoral efficacy in a syngeneic mouse tumor model. HPVp1 expressed higher amounts of HPV antigens compared to HPVp5 in vitro but had a slightly delayed replication kinetic which overall translated into increased HPV-specific T cell responses upon vaccination of mice. Immunization with both vectors protected mice in prophylactic and in therapeutic TC-1 tumor models with HPVp1 being more effective in the prophylactic setting. Taken together, VSV-GP is a promising candidate as therapeutic HPV vaccine and first position of the vaccine antigen in a VSV-derived vector seems to be superior to fifth position. 相似文献