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Mutants of nonproducer cell lines transformed by murine sarcoma virus. 3. Detection and characterization of RNA specific for helper and sarcoma viruses.

BALB/3T3 cells transformed by the Kirsten sarcoma virus (nonvirus producer BALB/3T3 cells) and mutant cell lines derived therefrom by treatment with bromodeoxyuridine (BrdU) were analyzed for expression of virus-specific RNA using single-stranded DNA transcripts of Rauscher leukemia virus (RLV), a virus activated in one of the cell lines (58-2T), and Ki-SV-specific DNA transcript; the latter transcript after removal of all sequences cross-reactive with RLV RNA. The Rauscher virus DNA detected multiple copies of viral RNA in virus-producing cells ( approximately 2.5 x 10(3)/cell) whether infected with RLV or activated to produce virus with BrdU. Nonproducer (NP) cells and normal BALB cells showed small numbers of RNA genomes (70-250/cell) and only partial saturation of the transcript. The intracellular RNA sedimented at 35S (main peak) with a variable minor peak at 20S with the exception of one mutant cell, M-43-2 (main peak at 26-27S). The 58-2T transcript reacted preferentially in NP cells and their derivatives with biphasic kinetics suggesting the possibility of sequences specific for the original transforming virus. The size of Ki-SV specific sequences were 30S in mutant cells whether or not complete virus was being produced and independent of in vivo transplantability.

Animals↗

Prognostic value of quantitative Kaposi sarcoma--associated herpesvirus load in posttransplantation Kaposi sarcoma.

Organ transplant recipients have a higher risk of Kaposi sarcoma (KS). A quantitative real-time polymerase chain reaction assay was developed to evaluate KS-associated herpesvirus (KSHV) as a prognostic tool in transplant recipients with KS. Forty-three patients who developed KS after transplantation were included in a cross-sectional study to correlate virus load with transplantation or KS parameters. Seventeen patients (40%) had KSHV viremia (>100 copies/microg of DNA; median, 6067 copies/microg of DNA). Factors associated with these levels of viremia by univariate analysis were progression of KS (P=.00002), time from KS diagnosis (P=.0007), actual stage of KS (P=.006), initial stage of KS (P=.22), graft loss (P=.013), and time from transplantation (P=.0246). Disease progression remained associated with KSHV viremia in a multivariate analysis (P=.01). Thus, quantification of KSHV load in peripheral blood mononuclear cells could represent a useful tool for monitoring transplant recipients with KS.

Cohort Studies↗

High incidence of Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus in tumor lesions and peripheral blood mononuclear cells from patients with Kaposi's sarcoma in Uganda.

With the advent of AIDS, Kaposi's sarcoma (KS) has become one of the leading malignancies in sub-Saharan Africa. Recently, DNA sequences from a new human herpesvirus called KS-associated herpesvirus (KSHV) or human herpesvirus type 8 have been found in KS tumor lesions in high frequency. Analyses of tumor lesions from 38 Ugandan KS patients indicated a uniform presence of KSHV in KS tumor lesions as revealed by polymerase chain reaction and Southern hybridization. In contrast, only 31% (11/36) of the normal skin biopsies from the same patient population were positive. The frequency of KSHV DNA detection in peripheral blood mononuclear cells (PBMC) of KS patients was also high (84%, 31/37). Similar analyses revealed the presence of cytomegalovirus (21% in KS lesions) to be discordant with KS development. A large number of KS lesions (87%, 33/38) and KS PBMC (70%, 26/37) were, however, positive for Epstein-Barr virus sequences. In addition, KSHV DNA was not found in the PBMC of Ugandans without KS.

Adult↗

AIDS presenting with cutaneous Kaposi's sarcoma and bacillary angiomatosis in the bone marrow mimicking Kaposi's sarcoma.

Kaposi's sarcoma (KS) and bacillary angiomatosis (BA) may be histologically similar. A precise diagnosis is required because of the different management of these diseases. KS or BA involving bone marrow is rare in patients with and without acquired immune deficiency syndrome (AIDS). We report the case of a 40-year-old human immunodeficiency virus (HIV)-positive homosexual male who presented with small KS lesions in the skin and BA in the bone marrow that histologically were similar. Laboratory evaluation revealed anemia and thrombocytopenia; CD4 count was 103/mm3, and the viral load was 750,000 HIV-1 mRNA copies per milliliter in plasma. Bartonella henselae, the etiologic agent of BA, was isolated from a blood culture. DNA sequences of human herpesvirus-8 (HHV-8), the putative etiologic agent of KS, were identified by polymerase chain reaction (PCR) in skin and bone marrow specimens, but antibody anti-HHV-8-encoded protein ORF73, localized signals only in the skin-KS lesion. The patient received clarithromycin and cefotetan for the BA, and antiretroviral therapy for the HIV infection. The skin lesions gradually regressed, the HIV-1 mRNA copy number decreased to less than 400 per milliliter and the CD4 lymphocyte count increased to 665/mm3. In conclusion, vascular lesions of BA and KS may be clinically and histologically similar, both may be associated with advanced AIDS, and an accurate diagnosis is required because of their different management.

AIDS-Related Opportunistic Infections↗

Detection of Kaposi's sarcoma-associated herpesvirus-like DNA sequence in vascular lesions. A reliable diagnostic marker for Kaposi's sarcoma.

A new herpesvirus-like DNA sequence (KSHV) has been recently identified in Kaposi's sarcoma (KS) from patients with AIDS and non-AIDS patients. To verify the specificity of the association of this new viral DNA with KS, a total of 155 cases of benign and malignant vascular neoplasms sharing similar histogenesis of endothelial derivation were analyzed for the presence of this KSHV sequence using the published 330-233 primers by polymerase chain reaction (PCR). The results revealed that all 17 cases of KS, both AIDS and non-AIDS, were positive for this KSHV, whereas the remaining 138 cases of vascular lesions other than KS, including 15 cases of angiosarcoma, showed negative reaction. These results confirm and extend the previous observation that this KSHV sequence is specifically associated with KS and is a reliable diagnostic marker to distinguish KS, particularly at its early stage, from other vascular lesions.

Acquired Immunodeficiency Syndrome↗

Combination chemotherapy with doxorubicin, dacarbazine, and ifosfamide in advanced adult soft tissue sarcoma. Canadian Sarcoma Group--National Cancer Institute of Canada Clinical Trials Group.

Forty-three adult patients with locally advanced or metastatic soft tissue sarcoma entered a pilot study of combination chemotherapy comprising 50 mg of doxorubicin/m2 by intravenous bolus, 850 mg of dacarbazine/m2 by 1-hour infusion, and 5 g of ifosfamide/m2 by 24-hour infusion with mesna uroprotection. The overall response rate in 40 assessable patients was 25% with two complete remissions. Twenty-four episodes of infection occurred in 148 courses (16%). These infections were usually associated with neutropenia (granulocyte count less than 0.5 X 10(9)/L), which occurred in 70% of the courses. These results do not differ from those elicited by each agent alone, and may reflect inadequacies of dose intensity or scheduling, or evaluation in a study population with adverse prognostic factors.

Adolescent↗

The evaluation of human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) in cutaneous lesions of Kaposi's sarcoma: a study of formalin-fixed paraffin-embedded tissue.

The etiologic role of human herpesvirus 8 (HHV8) in Kaposi's sarcoma (KS) remains controversial. This polymerase chain reaction (PCR)-based study was undertaken to evaluate the relationship of HHV8 in KS from our geographical region and to determine its utility in formalin-fixed, paraffin-embedded biopsy specimens. Forty-two biopsy specimens were studied, including nine AIDS-associated cases and four from classic KS. Of the 39 patients included, 21 were HIV+ and 18 had no clinical evidence of HIV infection. Only a single (internal) primer pair was used in the PCR. All PCR products were tested with an HHV8 specific probe by dot blot analysis. Seven of the nine AIDS KS specimens were positive for HHV8 on electrophoresis, and all nine were positive after dot blot. Two of four specimens from classic KS were positive for HHV8 on the gel electrophoresis, and three of four were positive on blot confirmation. Non-KS specimens from 15 HIV+ patients yielded one positive venous hemangioma from a man with HHV8-positive KS at another site. Specimens from HIV-unrelated controls with a variety of diagnoses were negative by PCR with blot. The predominant negativity for HHV8 in non-KS lesions suggests that HHV8 is not widespread in our patients. These results demonstrate yet another series linking KS with HHV8 and show that paraffin-embedded biopsy specimens are satisfactory for this technique.

Adolescent↗

Kaposi's sarcoma-associated herpesvirus infection prior to onset of Kaposi's sarcoma.

OBJECTIVES: Kaposi's sarcoma-associated herpesvirus (KSHV), a newly discovered human gammaherpesvirus, is found in the majority of KS lesions from patients with and without AIDS. Peripheral blood mononuclear cells (PBMC) were examined for KSHV DNA to determine whether viral infection precedes onset of this neoplasm. DESIGN: Randomized and blinded case-control study of prospectively collected PBMC samples from ongoing cohort studies. METHODS: Paired PBMC drawn before and after KS onset from 21 AIDS-KS patients were compared to paired PBMC from 23 high-risk HIV-infected homo-/bisexual patients who did not develop KS and to a single PBMC sample from 19 low-risk, HIV-infected hemophiliac patients. Extracted DNA samples were amplified by polymerase chain reaction (PCR) using two non-overlapping nested primer sets to control for potential PCR contamination. RESULTS: In all comparisons, patients who went on to develop KS were significantly more likely to show evidence of KSHV infection prior to onset of KS than either control group. Of PBMC samples from AIDS-KS patients drawn prior to KS, 52% were positive for KSHV DNA whereas both high- and low-risk control groups had lower rates of PBMC infection (9-13%). KSHV infection can precede KS onset by up to 21 months among AIDS-KS patients. CONCLUSIONS: AIDS-KS patients are significantly more likely to show evidence of KSHV infection in PBMC prior to KS onset than control HIV-infected patients. Because identical PBMC samples from cases and controls were examined blindly, these results are not caused by a bias in tissue sampling. Homo-/bisexual and hemophiliac AIDS patients who do not develop KS appear to have a low prevalence of infection. These findings indicate that KSHV infection is specifically associated with the subsequent development of KS in AIDS patients.

AIDS-Related Opportunistic Infections↗

The beta-core fragment of human chorionic gonadotrophin inhibits growth of Kaposi's sarcoma-derived cells and a new immortalized Kaposi's sarcoma cell line.

OBJECTIVE: Kaposi's sarcoma (KS), a condition often associated with HIV infection, is more common in men than in women; pregnancy and sex hormones could be involved. Urinary human chorionic gonadotrophin (hCG) has been reported to inhibit the growth of KS cell lines, with great variability among preparations. Urinary hCG often contains free forms of the hCG subunits and a fragment of the free beta-subunit, the beta-core, which may have biological activity. We compared the effect of the beta-core fragment, the beta-subunit, recombinant and urinary hCG on KS immortal and spindle cells. DESIGN AND METHODS: A new immortal KS cell line was phenotypically and karyotypically characterized. The effects on growth of this cell line and of primary KS spindle cells by hCG and its purified derivatives were tested. Induction of apoptosis was demonstrated using acridine orange/ethidium bromide staining. RESULTS: The beta-core fragment harboured the most potent growth inhibitory activity on a molar basis. After 72 h of treatment with the beta-core, 60-70% of KS cells show apoptotic nuclei. No effects were observed on endothelial cells. CONCLUSIONS: The beta-core fragment of hCG proved to be the most effective part of the hCG molecule, inducing growth inhibition and apoptosis of KS cells. Thus, the beta-core could be the most appropriate hCG derivative for the therapy of KS.

Antineoplastic Agents↗

Prospective study of the effects of antiretroviral therapy on Kaposi sarcoma--associated herpesvirus infection in patients with and without Kaposi sarcoma.

OBJECTIVE: To investigate the effect of highly active antiretroviral therapy (HAART) on circulating Kaposi sarcoma (KS)-associated herpesvirus (KSHV) load in HIV-infected individuals with and without KS. DESIGN: Twenty-nine HIV-infected subjects (21 with KS and 8 without KS) were recruited for the study; they were prospectively studied before and at regular intervals during HAART. METHODS: Patients with KS were clinically assessed using Adult Clinical Trials Group (ACTG) criteria, and sequential blood samples were obtained from all patients for determination of plasma HIV-1 load, CD4 cell count, titer of antibody to KSHV, and KSHV load. RESULTS: Ten of 21 patients with KS had a favorable KS response (complete response, 6; partial response, 4) with HAART alone. Of the 20 subjects with detectable KSHV viremia prior to HAART, over one half (12 [60%]) had an undetectable KSHV load with antiretroviral therapy. There was no significant difference between subjects receiving protease inhibitor- or nonnucleoside reverse transcriptase inhibitor-based treatment combinations. Achieving undetectable KSHV viremia is associated with a better clinical outcome in patients with KS. CONCLUSIONS: To our knowledge, we demonstrate for the first time that both protease inhibitor-based and nonnucleoside reverse transcriptase inhibitor-based antiretroviral treatment combinations may lead to an undetectable KSHV load and confirm that an undetectable KSHV load is associated with KS regression. There was no clear association between CD4 cell count response and KS response to HAART, but there was a significant relationship between HIV load response to HAART and clinical improvement of KS.

Adult↗

Human herpesvirus 8/Kaposi sarcoma herpesvirus cell association during evolution of Kaposi sarcoma.

Kaposi sarcoma (KS) is associated with a herpesvirus (HHV-8/KSHV), which expresses a latency-associated nuclear antigen (LANA). The histopathology of KS is characterized by angiogenesis, inflammatory cells, and the development of CD34+ tumor spindle cells (SCs). However, the cellular basis for the recruitment and dissemination of HHV-8 during the development of KS lesions is not clear. Twenty-nine KS biopsies with AIDS (AKS, n=22) and without HIV infection (endemic KS or EKS, n=7) were immunostained by a triple antibody method to characterize HHV-8-infected and noninfected (LANA+/-) CD34+ SCs, infiltrating CD3+, CD68+, CD20+, and CD45+ leukocytes as well as proliferating (Ki67+) cells. The CD34+/LANA+ SCs were more frequent in late (nodular) as compared with early (patch/plaque) KS stages. However, in late AKS 36.0% of SCs (median of 11 cases) were CD34+/LANA- compared with 20.7% in early cases (median of 11 cases). Furthermore, both AKS and EKS showed, at all stages, a small (4.1-6.5%) population of LANA+/CD34- cells. Proliferating Ki67+ cells were seen (4.5-11.5%) at all KS stages, and were usually more frequent in early AKS, but no significant difference was observed between nodular AKS and EKS. Most of the proliferating cells in the KS lesions were LANA+/CD34+ but a small fraction was LANA+/CD34-. Lesional CD68+ and CD3+ cells varied between AKS (7.3 and 5.2%, respectively) and EKS (4.9 and 3.1%, respectively) but were not clearly stage related. No LANA+ cells were CD3+, CD20+, or CD45+ and very few (<0.5%) were CD68+. These results indicate that not all CD34+ KS SCs were LANA+, suggesting recruitment of noninfected SCs to the lesions. Cell proliferation in general was much higher in early as compared with the late AKS stages. LANA+ SCs could have a proliferative advantage as suggested by higher frequency of cycling (Ki67+) LANA+ SCs. Few macrophages but no lymphocytes are LANA+.

Acquired Immunodeficiency Syndrome↗

Natural killer cells are not infected by Kaposi's sarcoma-associated herpesvirus in vivo, and natural killer cell counts do not correlate with the risk of developing Kaposi's sarcoma.

Although the innate immune system is implicated in the control of Kaposi's sarcoma (KS), the risk of developing KS is not associated with the nadir natural killer (NK) cell count, and NK cell counts do not significantly increase or decrease during KS resolution. KS-associated herpesvirus replication was not demonstrated in vivo or in vitro within NK cells, suggesting that NK cells do not contribute to the resolution of KS. Their role appears limited to events occurring during early infection.

Antiretroviral Therapy, Highly Active↗

Neuronal differentiation of Ewing's sarcoma induced by cholera toxin B and bromodeoxyuridine--establishment of Ewing's sarcoma cell line and histochemical study.

An Ewing's sarcoma (ES) cell line was established from a metastatic bone marrow specimen in a patient with advanced disease, and some histochemical characteristics were investigated by neuronal differentiation induced with cholera toxin B (CTB) and bromodeoxyuridine (BrdU). Neuronal differentiation was investigated by the expression of neurofilament and Leu-7, and glial differentiation was observed by expression of S-100 protein. Neurofilament (NF) and Leu-7 were positive in ES cells and these were expressed more intensively by induction with CTB than with BrdU. There was no expression of S-100 protein in untreated or differentiated ES cells. ES cells became differentiated to neuronal cells with CTB and BrdU, but it was not observed, that ES cells had the potential to differentiate to glial cells. It appears that ES is of more primitive neural origin than neuroblastoma, primitive neuroectodermal tumors and other related neural tumors.

Antigens, Differentiation↗

Rescue of rous sarcoma virus from rous sarcoma virus-transformed mammalian cells.

Rat cells transformed by the B77 strain of avian sarcoma virus produce no virus-like particles, yet B77 virus was rescued from these cells by Sendai virus-mediated fusion with chicken cells. This virus rescue was not affected by treatment of the chicken cells with agents that rendered the cells incapable of dividing, although such treatment greatly reduced the ability of the chicken cells to plate as infectious centers after infection with B77 virus. Fusion of R(B77) cells with chicken erythrocytes also led to virus rescue, although with less efficiency than fusion with chicken fibroblasts. Therefore, virus rescue was probably due to a factor or factors contributed by chicken cells which aid in virus production.

Animals↗

Relationship between Moloney murine leukemia and sarcoma virus RNAs: purification and hybridization map of complementary DNAs from defined regions of Moloney murine sarcoma virus 124.

Complementary DNAs (cDNA's) specific for various regions of the Moloney murine sarcoma virus (MSV) 124 RNA genome were prepared by cross-hybridization techniques. A cDNA specific for the first 1,000 nucleotides adjacent to the RNA 3' end (cDNA 3') was prepared and shown to also be complementary to the 3'-terminal 1,000 nucleotides of a related Moloney murine leukemia virus (MLV) genome. A cDNA complementary to the "MSV-specific" portion of the MSV 124 genome was prepared. This cDNA was shown not to anneal to Moloney MLV RNA and to anneal to a portion of the viral RNA of about 1,500 to 1,800 nucleotides in length, located 1,000 nucleotides from the 3' end of MSV RNA. A cDNA common to the genome of MSV and MLV was also obtained and shown to anneal to the 5'-terminal two-thirds, as well as to the 3'-terminal 1,000 nucleotides, of the MSV RNA genome. This cDNA also annealed to the RNA from MLV and mainly to the 5'-terminal half of the MLV genome. It is concluded that the 6-kilobase Moloney MSV 124 RNA genome has a sequence arrangement that includes (i) a 3' portion of about 1,000 nucleotides, which is also present at the 3' terminus of MLV; (ii) an MSV-specific region, not shared with MLV, which extends between 1,000 and 2,500 nucleotides from the 3' terminus; and (iii) a second "common" region, again shared with MLV, which extends from 2,500 nucleotides to the 5' terminus. This second common region appears to be located in the 5' half of the 10-kilobase MLV genome as well. Experiments in which a large excess of cold MLV cDNA was annealed to (3)H-labeled polyadenylic acid-containing fragments of MSV RNA gave results consistent with this arrangement of the MSV genome.

Base Sequence↗

Detection of the viral sarcoma gene product in cells infected with various strains of avian sarcoma virus and of a related protein in uninfected chicken cells.

Genetic analyses have defined a single gene (src) as that portion of the avian sarcoma virus (ASV) genome which encodes the protein directly responsible for ASV-induced neoplastic transformation. We have recently identified the polypeptide product of the src gene of the Schmidt-Ruppin (SR) strain of ASV, a 60,000-dalton phosphoprotein designated pp60(src), and have further determined that pp60(src) acts as a protein kinase. Essential to the identification and characterization of the pp60(src) protein of SR-ASV was the use of serum (TBR serum) from rabbits bearing SR-ASV-induced tumors. TBR serum was, however, strain specific, recognizing pp60(src) from SR-ASV-transformed cells only. We report here that sera from marmosets bearing tumors induced by the Bryan or SR strains of ASV (TBM sera) contain antibody which precipitates the transforming gene product from cells transformed by the SR, Bryan, Prague, or Bratislava strains of ASV. In contrast, rabbits bearing tumors induced by either the Bratislava or Bryan strains of ASV, or hamsters with SR-ASV-induced tumors did not produce antibody to pp60(src) from any strain of ASV. The 60,000-dalton polypeptides immunoprecipitated with TBM serum from cells transformed by each of the above virus strains are phosphoproteins. One-dimensional peptide mapping by limited proteolysis revealed that the pp60(src) proteins are structurally very similar, but not identical. Furthermore, all of the viral pp60(src) proteins have an associated phosphotransferase activity. In addition to detecting the viral src proteins, TBM serum was able to immunoprecipitate an antigenically related protein from normal uninfected avian cells.

Animals↗

Inheritance of susceptibility to the myeloproliferative sarcoma virus: effect of the Fv-2 locus and evidence for a myeloproliferative sarcoma virus resistance locus.

Myeloproliferative sarcoma virus (MPSV) causes a generalized stem cell leukemia with erythroid and myeloid hyperplasia in adult mice. MPSV also transforms fibroblasts. Mice congenic for the Fv-2 locus showed marked differences in susceptibility to MPSV according to the Fv-2 genotype. MPSV was injected into C57BL/6 Fvs and C57BL/6 Fv-2r mice congenic except for the Fv-2 locus. C57BL/6 mice with the Fvs genotype were much more susceptible to MPSV than were those with the Fvr genotype. Both DDD Fv-2r mice congenic with DDD Fv-2s mice except for the Fv-2 locus and DDD Fv-2s mice, however, were sensitive to spleen focus formation by MPSV. These data indicate that at least one additional resistance locus to MPSV is present in C57BL/6 mice but not in DDD mice. Both the Fv-2 locus and the putative MSPV resistance locus (loci) Mpsvr appear to be epistatic to either of the sensitivity loci. Fibroblast focus formation by MPSV was obtained well in C57BL/6 Fv-2r and C57BL/6 Fvs fibroblasts, indicating that the genes for MPSV resistance (Fv-2r and Mpsvr) were not operating in fibroblast cells. A model is proposed which may account for the differences in response of genetically different mice to MPSV and Friend spleen focus-forming virus.

Alleles↗

Translational products of Moloney murine sarcoma virus RNA: identification of proteins encoded by the murine sarcoma virus src gene.

In vitro translation of virion RNA of Moloney murine sarcoma virus (MSV) strain 124 yielded major products having molecular weights of 63,000 (63K), 43K, 40K, 31K, and 24K daltons. A molecularly cloned subgenomic fragment of Moloney MSV comprised of the cellular insertion (src) region was utilized in hybridization arrest translation as a means of identifying products of the MSV src gene. MSV src DNA specifically inhibited synthesis of the 43K, 40K, 31K, and 24K proteins, implying that each of these proteins was coded within the MSV src gene. The MSV src-specific nature of this family of proteins was further confirmed by partial purification of MSV src-containing RNAs from MSV non-producer cells. In vitro translation of enriched cellular RNAs yielded products with molecular weights identical to those of the 43K family of proteins synthesized from virion RNA. Nucleotide sequence analysis of the MSV transforming region has revealed a long open reading frame which includes five methionine codons (Reddy et al., Proc. Natl. Acad. Sci. U.S.A. 77:5234-5238, 1980). The molecular weights of the four largest proteins that could be synthesized within this open reading frame corresponded closely to the molecular weights of the 43K family of proteins. Partial cyanogen bromide cleavage of each of the three largest proteins resulted in an uncleaved fragment having a molecular weight equal to that of the smallest (24K) protein. These findings provide direct biochemical evidence that the 43K, 40K, 31K, and 24K proteins are related in their carboxy-terminal regions, as well as information concerning the MSV src gene coding sequences from which each protein originates:

Animals↗