Activation of endogenous retroviral sequences in human leukemia

Endogenous retroviral sequences have been identified in the genomes of several species including humans. Proteins similar to those of primate endogenous viruses have been found on the surface of various malignant cells in man. To further define this role, we have used a primate retrovirus DNA from the Baboon Endogenous Virus to probe a human genomic library for related sequences. A total of 45 clones homologous to BaEV gag-pol were isolated under low stringency hybridization. Of these, several were found to contain DNA which was expressed as RNA at higher levels in human lymphoid leukemic cells than in normal lymphocytes.     

Reliability of the RNA-DNA Filter Hybridization for the Detection of Oncornavirus-Specific DNA Sequences

Denatured DNA from leukemic myeloblasts or uninfected chicken embryos, immobilized on nitrocellulose filters, was hybridized to a vast excess of [(3)H]70S RNA from purified avian myeloblastosis virus. The viral RNA was eluted from the RNA-DNA hybrids, purified, and then rehybridized in solution to an excess of either leukemic or normal chicken embryonic DNA. This study revealed that all the slow and the fast hybridizing viral RNA sequences detectable by liquid hybridization in DNA excess had hybridized to the filter bound DNA. Both techniques also gave similar values for the number of 28S ribosomal RNA genes contained in a chicken cell genome: 210 by the liquid hybridization procedure and 218 by the filter hybridization technique. Therefore, filter hybridization can accurately detect DNA sequences present in relatively few numbers in the genome of higher organisms.     

Expression of Endogenous Retroviral Genes in Leukemic Guinea Pig Cells

The expression of guinea pig retrovirus (5-bromodeoxyuridine[BUdR]-induced GPV) was studied in guinea pig L(2)C leukemic lymphoblasts by use of molecular hybridization of viral complementary DNA (cDNA) to cellular RNA. It was found that L(2)C leukemic lymphoblasts, leukemic spleen, and BUdR-induced virus-producing cells contain virus-specific RNA: 0.05% (800 to 960 copies per cell), 0.02% (360 copies per cell), and 0.3% (5,120 copies per cell), respectively. Adult normal liver and spleen, on the other hand, contain less than 0.2 copy of viral RNA per cell. Both BUdR-induced cells and L(2)C leukemic lymphoblasts contained 14S, 22S, 35S, and 70S RNA species of total and cytoplasmic virus-specific RNA as determined by sucrose velocity gradient analysis and hybridization of sucrose gradient fractions to cDNA. Virus-specific mRNA was identified in both BUdR-induced cells and L(2)C leukemic lymphoblasts by the criterion that it cosedimented with purified polyribosomes in a sucrose gradient and that it changed to a lower sedimentation value if polyribosomes were disaggregated with EDTA prior to centrifugation. Virus-specific mRNA obtained from either the polyribosome region of purified polyribosomes or the released messenger region of EDTA-disaggregated purified polyribosomes consisted of 14S, 20S, and 35S species in both BUdR-induced cells and L(2)C leukemic lymphoblasts. Hybridization of cDNA to the RNA of L(2)C leukemic lymphoblasts and BUdR-induced cells was essentially complete. Additionally, leukemic lymphoblast RNA could displace 95% of the hybridization of BUdR-induced GPV 70S RNA to guinea pig DNA. The midpoints of thermal denaturation of hybrids formed between GPV cDNA and the RNA of either L(2)C leukemic lymphoblasts or the 70S RNA of BUdR-induced GPV were both 89 degrees C in 2x concentrated 0.15 M NaCl plus 0.015 M sodium citrate. These results show that BUdR-induced GPV genes are essentially completely expressed in L(2)C leukemic lymphoblasts and that virus-specific mRNA is present, although fewer copies of RNA are present in L(2)C leukemic lymphoblasts than in BUdR-induced cells.     

Ribonucleotide Sequence Homology Among Avian Oncornaviruses

RNA sequence relatedness among avian RNA tumor virus genomes was analyzed by inhibition of DNA-RNA hybrid formation between ³H-labeled 35S viral RNA and an excess of leukemic or normal chicken cell DNA with increasing concentrations of unlabeled 35S viral RNA. The avian viruses tested were Rous associated virus (RAV)-0, avian myeloblastosis virus (AMV), RAV-60, RAV-61, and B-77 sarcoma virus. Hybridization of ³H-labeled 35S AMV RNA with DNA from normal chicken cells was inhibited by unlabeled 35S RAV-0 RNA as efficiently (100%) as by unlabeled AMV RNA. Hybridization between ³H-labeled 35S AMV RNA and DNA from leukemic chicken myeloblasts induced by AMV was suppressed 100 and 68% by unlabeled 35S RNA from AMV and RAV-0, respectively. Hybridization between ³H-labeled RAV-0 and leukemic chicken myeloblast DNA was inhibited 100 and 67% by unlabeled 35S RNA from RAV-0 and AMV, respectively. It appears therefore that the AMV and RAV-0 genomes are 67 to 70% homologous and that AMV hybridizes to RAV-0 like sequences in normal chicken DNA. Hybridization between AMV RNA and leukemic chicken DNA was inhibited 40% by RNA from RAV-60 or RAV-61 and 50% by B-77 RNA. Hybridization between RAV-0 RNA and leukemic chicken DNA was inhibited 80% by RAV-60 or RAV-61 and 70% by B-77 RNA. Hybridization between ³H-labeled 35S RNA from RAV-60 or RAV-61 and leukemic chicken myeloblast DNA was reduced equally by RNA from RAV-60, RAV-61, AMV or RAV-0; this suggests that RNA from RAV-60 and RAV-61 hybridizes with virus-specific sequences in leukemic DNA which are shared by AMV, RAV-0, RAV-60, and RAV-61 RNAs. Hybridization between ³H-labeled 35S RNA from RAV-61 and normal pheasant DNA was inhibited 100% by homologous viral RNA, 22 to 26% by RNA from AMV or RAV-0, and 30 to 33% by RNA from RAV-60 or B-77. Nearly complete inhibition of hybridization between RAV-0 RNA and leukemic chicken DNA by a mixture of AMV and B-77 35S RNAs indicates that the RNA sequences shared by B-77 virus and RAV-0 are different from the sequences shared by AMV and RAV-0. It appears that different avian RNA tumor virus genomes have from 50 to 80% homology in nucleotide sequences and that the degree of hybridization between normal chicken cell DNA and a given viral RNA can be predicted from the homology that exists between the viral RNA tested and RAV-0 RNA.     

Acquisition of new DNA sequences after infection of chicken cells with avian myeloblastosis virus

DNA-RNA hybridization studies between 70S RNA from avian myeloblastosis virus (AMV) and an excess of DNA from (i) AMV-induced leukemic chicken myeloblasts or (ii) a mixture of normal and of congenitally infected K-137 chicken embryos producing avian leukosis viruses revealed the presence of fast- and slow-hybridizing virus-specific DNA sequences. However, the leukemic cells contained twice the level of AMV-specific DNA sequences observed in normal chicken embryonic cells. The fast-reacting sequences were two to three times more numerous in leukemic DNA than in DNA from the mixed embryos. The slow-reacting sequences had a reiteration frequency of approximately 9 and 6, in the two respective systems. Both the fast- and the slow-reacting DNA sequences in leukemic cells exhibited a higher T_m (2 C) than the respective DNA sequences in normal cells. In normal and leukemic cells the slow hybrid sequences appeared to have a T_m which was 2 C higher than that of the fast hybrid sequences. Individual non-virus-producing chicken embryos, either group-specific antigen positive or negative, contained 40 to 100 copies of the fast sequences and 2 to 6 copies of the slowly hybridizing sequences per cell genome. Normal rat cells did not contain DNA that hybridized with AMV RNA, whereas non-virus-producing rat cells transformed by B-77 avian sarcoma virus contained only the slowly reacting sequences. The results demonstrate that leukemic cells transformed by AMV contain new AMV-specific DNA sequences which were not present before infection.     

Ribonucleotide sequence homology among avian oncornaviruses.

RNA sequence relatedness among avian RNA tumor virus genomes was analyzed by inhibition of DNA-RNA hybrid formation between 3H-labeled 35S viral RNA and an excess of leukemic or normal chicken cell DNA with increasing concentrations of unlabeled 35S viral RNA. The avian viruses tested were Rous associated virus (RAV)-3, avian myeloblastosis virus (AMV), RAV-60, RAV-61, and B-77 sarcoma virus. Hybridization of 3H-labeled 35S AMV RNA with DNA from normal chicken cells was inhibited by unlabeled 35S RAV-0 RNA as effeciently (100%) as by unlabeled AMV RNA. Hybridization between 3H-labeled 35S AMV RNA and DNA from leukemic chicken myeloblasts induced by AMV was suppressed 100 and 68% by unlabeled 35S RNA from AMV and RAV-0, respectively. Hybridization between 3H-labeled RAV-0 and leukemic chicken myeloblast DNA was inhibited 100 and 67% by unlabeled 35S RNA from RAV-0 and AMV, respectively. It appears therefore that the AMV and RAV-0 genomes are 67 to 70% homologous and that AMV hybridizes to RAV-0 like sequences in normal chicken DNA. Hybridization between AMV RNA and leukemic chicken DNA was inhibited 40% by RNA from RAV-60 or RAV-61 and 50% by B-77 RNA. Hybridization between RAV-0 RNA and leukemic chicken DNA was inhibited 80% by RAV-60 or RAV-61 and 70% by B-77 RNA. Hybridization between 3H-labeled 35S RNA from RAV-60 or RAV-61 and leukemic chicken myeloblast DNA was reduced equally by RNA from RAV-60, RAV-61, AMV or RAV-0; this suggests that RNA from RAV-60 and RAV-61 hybridizes with virus-specific sequences in leukemic DNA which are shared by AMV, RAV-0, RAV-60, and RAV-61 RNA'S. Hybridization between 3H-labeled 35S RNA from RAV-61 and normal pheasant DNA was inhibited 100% by homologous viral RNA, 22 TO 26% BY RNA from AMV or RAV-0, and 30 to 33% by RNA from RAV-60 or B-77. Nearly complete inhibition of hybricization between RAV-0 RNA and leukemic chicken DNA by a mixture of AMV and B-77 35S RNAs indicates that the RNA sequences shared by B-77 virus and RAV-0. It appears that different avian RNA tumor virus genomes have from 50 to 80% homology in nucleotide sequences and that the degree of hybridization between normal chicken cell DNA and a given viral RNA can be predicted from the homology that exists between the viral RNA tested and RAV-0 RNA.     

Inhibition of DNA polymerase alpha from leukemic and normal human cells by partially thiolated human deoxyribonucleic acids

In continuing search for exploitable biochemical differences between cancer and normal cells at the level of DNA replication, leukemic and "normal" hematopoietic cells from four different, established human cell lines were grown in culture flasks, and both the DNA and the DNA polymerase alpha were isolated in each case from the harvested (5-10 g wet weight) cell pellets. The four selected cell lines included a "normal" lymphoblastoid B-cell line (RPMI-1788), a pre-B cell (NALM-6) and a T-cell (MOLT-4) acute lymphoblastic leukemias, and a promyelocytic leukemia (HL-60). The DNA polymerase alpha enzyme of the two B-cell lines (both the leukemic and the "normal") showed the usual sensitivity toward inhibition by aphidicolin, while those from the two other leukemic cell lines were remarkably resistant to the antibiotic. Partially thiolated polycytidylic acid (MPC) strongly inhibited only the DNA polymerase alpha of the "normal" cell line, whereas the corresponding enzymes of all three leukemic cell lines were relatively insensitive to MPC. In contrast, the partially thiolated DNAs derived from the leukemic cell lines more strongly inhibited the DNA polymerase alphas of the leukemic cell lines than that of the "normal" cell line. These results indicate the existence of some structural differences between the DNA polymerase alpha enzymes (as well as between the DNAs) of human cells of different lineage and, particularly, of leukemic vs. "normal" character; such differences could be exploited in the design of selective antitemplates for chemotherapy.     

Cloning of the human oestrogen receptor cDNA

Poly A+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and those fractions enriched in oestrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt11 vectors. Clones corresponding to the ER were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kbase) and lambda OR8 (2.1 kbase), isolated using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains DNA sequences which cross-hybridize with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Using lambda OR8 as a hybridization probe revealed a single poly A+ RNA band of approx. 6.2 kbase in the ER containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-cell line HeLa. The same probe hybridizes to a chicken gene which is expressed in oviduct tissue as a 7.5 kbase poly A+ RNA.     

Human leukemic cells: Biological action of exogenous human leukemic DNA

Intact exogenous human leukemie DNA derived from cells in culture was taken up by both normal and leukemic recipient human cells, wherein it migrates to the nucleus and becomes associated with host genome. Uptake of exogenous DNA averages about 15–20 percent and was relatively higher in leukemic than in normal cells in a given culture medium.Isologous and homologous human leukemic cells were more sensitive to inhibition by this exogenous DNA than were normal human cells. Both DNA and RNA synthesis were inhibited, but protein synthesis was stimulated — effects similar to those described consequent to exposure to certain viruses.Immunological studies of hamster cells treated with human leukemic DNA failed to show any presence of human surface antigens. The in vivo studies showed that this metabolically active radioactive DNA had migrated to several organs of hamsters and gerbils, the highest labeled DNA activity being found in the testis and kidney of these animals.Prolonged exposure to exogenous leukemic DNA resulted in marked phenotypic changes in normal human fibroblasts, which thus far appear to be heritable. Search for evidence of genotypic changes in these altered cells which might relate these observations to «neoplastic transformation is in progress.     

Acquisition of viral DNA sequences in target organs of chickens infected with avian myeloblastosis virus.

The distribution of oncornavirus DNA sequences in various tissues of normal chickens and of chickens with leukemia or kidney tumors induced by avian myeloblastosis virus (AMV) was analyzed by DNA-RNA hybridization using 35S AMV RNA as a probe. All the tissues from normal chickens which were tested contained the same average cellular concentration of endogenous oncornavirus DNA. In contrast, different tissues from lekemic chickens and from chickens bearing kidney tumors contained different concentrations of AMV homologous DNA: in some tissues there was no increase whereas other tissues acquired additional AMV-specific DNA sequences. The increase was the greatest in tissues which can become neoplastic after infection, such as myeloblasts, erythrocytes, and kidney cells. It was directly demonstrated that DNA from AMV-induced kidney tumor contains AMV sequences which are absent in DNA from normal cells. A similar finding had been previously obtained with leukemic cells (15). 3H-labeled 35S RNA from purified AMV was exhaustively hybridized with an excess of normal chicken DNA to remove all the viral RNA sequences which are complementary to DNA from uninfected cells. The 3H-labeled RNA which failed to hybridize was isolated by hydroxylapatite column chromatography which separates DNA-RNA hybrids from single-stranded RNA. The residual RNA hybridized to chicken kidney tumor DNA but did not rehybridize with normal chicken DNA.     

Integrated State of Oncornavirus DNA in Normal Chicken Cells and in Cells Transformed by Avian Myeloblastosis Virus

The covalent linkage of oncornavirus-specific DNA to chicken DNA was investigated in normal chicken embryo fibroblasts (CEF) and in virus-producing leukemic cells transformed by avian myeloblastosis virus (AMV). The virus-specific sequences present in cellular DNA fractionated by different methods were detected by DNA-RNA hybridization by using 70S AMV RNA as a probe. In CEF and in leukemic cells, the viral DNA appeared to be present only in the nucleus. After cesium chloride-ethidium bromide density equilibrium sedimentation, the viral DNA was present as linear, double-stranded molecules not separable from linear chicken DNA. After extraction by the Hirt procedure, the viral DNA precipitated with the high-molecular-weight DNA. After alkaline sucrose velocity sedimentation, the viral DNA cosedimented with the high-molecular-weight cellular DNA. The results indicate that in both types of cells studied, the oncornavirus-specific DNA sequences were linked by alkali stable bonds to nuclear cellular DNA of high molecular weight and did not appear to be present in free form of any size.     

Identification and characterization of estrogen-regulated RNAs in human breast cancer cells

Two cDNA libraries have been constructed with RNA prepared from the estrogen-responsive breast cancer cell lines, MCF7 and ZR 75. They were screened by differential hybridization for estrogen-regulated sequences. A total of 11 different RNAs were isolated from the MCF7 cell cDNA library and four from the ZR 75 cell cDNA library. Only two sequences were isolated from both libraries. The levels of the 13 different RNAs are induced between 2.5- and 100-fold by estrogen in MCF7 cells. The expression and regulation by estrogen of the RNAs was examined in eight different human tumor cell lines. The relative abundance of each RNA varied in the different cell lines. The expression of three RNAs (pNR-1, pNR-2, and pNR-25) was detected only in estrogen-responsive breast cancer cells. The sequences that were expressed in all eight cell lines were regulated by estrogen only in the three estrogen-responsive breast cancer cell lines. The response of the RNAs to other classes of steroids and to different concentrations of estrogen was characterized in more detail. The extent to which different concentrations of estradiol induced each RNA varied, but half-maximal induction of most of the RNAs occurred between 2 and 5 X 10(-11) M. The time at which increased RNA levels were first detected following exposure to estradiol also varied. Estrogen increased the levels of some RNAs within 15 min, while for others there was a lag of 4 h.     

Isolation and identification of lymphocytic and myelogenous leukemia-specific sequences in genomes of gibbon oncornaviruses

Five gibbon ape leukemia virus substrains (two from gibbons with lymphocytic leukemia and three from gibbons with myelogenous leukemia) were examined for unique genomic sequences specific for each form of leukemia. By using sequential adsorption procedures, the genome from each gibbon ape leukemia virus was fractionated into four sets of distinct nucleotide sequences. Based on their hybridization specificities toward DNAs of leukemic tissues, these sequences were designated as follows: (i) “COM,” (ii) “LYM” or “MYE,” (iii) “UNI,” and (iv) “UND.” The COM fraction represented sequences common to all of the viral genomes. The LYM fraction, which was isolated only from gibbon ape leukemia viruses associated with lymphocytic leukemia, represented genomic sequences associated with lymphocytic leukemia since the RNA hybridized at a 4- to 15-fold-higher rate to infected tissue DNA from lymphocytic leukemic gibbons than to infected tissue DNA from myelogenous leukemic gibbons. The MYE fraction, which was isolated only from gibbon ape leukemia viruses associated with myelogenous leukemia, represented genomic sequences associated with myelogenous leukemia since the RNA hybridized at a 5- to 15-fold-higher rate to infected tissue DNA from myelogenous leukemic gibbons than to infected tissue DNA from lymphocytic leukemic gibbons. The UNI fraction contained sequences unique to one virus substrain. The UND fraction contained sequences which varied depending upon the substrains involved in the adsorption procedures. These findings suggest that each gibbon ape leukemia virus examined in this study contains subgenomic sequences that are specifically identifiable only with the form of leukemia from which the virus was isolated.