Sequences Present in Both Human Leukemic Cell Nuclear DNA and Rauscher Leukemia Virus

DNA synthesized by particulate fractions from human leukemic white blood cells has been subfractionated by hybridization to Rauscher leukemia virus 70S RNA followed by hydroxylapatite chromatography. The Rauscher-leukemia-virus-specific DNA fraction is shown to be complementary only to sequences present in the nuclear DNA of Balb/c mouse spleens infected by this virus and the nuclear DNA from human leukemic white blood cells, and not to sequences present in the nuclear DNA of normal Balb/c mouse spleens or the nuclear DNA of normal human white blood cells.     

Crossreactive Antigens on Human Cells Infected with Rauscher Leukemia Virus and on Human Acute Leukemia Cells

A water-soluble component of membranes of cells from a Burkitt's lymphoma cultured cell line was used to develop an antiserum that detects an antigen(s) on acute leukemia cells. This antiserum did not react with nonlymphoid cultured cell lines except for human embryonic kidney cells infected with the Rauscher murine leukemia virus. Absorption studies demonstrated this antigen to be crossreactive with the antigen detected on the human acute leukemia cells.     

RNA in Human Leukemic Cells Related to the RNA of a Mouse Leukemia Virus

Molecular hybridization with radioactively labeled DNA complementary to the RNA of the Rauscher leukemia virus was used to probe for homologous RNA in the polysome fraction of human leukemic cells. The leukocytes of 24 out of 27 patients examined contained RNA possessing homology to that of the mouse leukemia agent, but not to that of the unrelated viruses causing mammary tumors in mice or myeloblastosis in chickens. Further, no control human leukocytes or other adult and fetal tissues showed significant levels of the leukemia-specific RNA. It would appear that human leukemic cells contain RNA sequences homologous to those found in a viral agent known to cause leukemia in an experimental animal. The fact that human sarcomas have also been shown to contain this type of RNA points to a remarkable parallelism in the leukemias and sarcomas of mice and men.     

Responsiveness of Human Granulocytic Leukemic Cells to Colony-stimulating Factor

The proliferative response of normal andleukemic human granulocytic cells to stimulation by varying concentrations of thenormal regulator, colony-stimulating factor (CSF), was determined by clustercounts in agar cultures of 150 blood ormarrow specimens stimulated by monkeylung conditioned medium. Acute myeloidleukemic cells were slightly more responsive than normal at low concentrations of conditioned medium, but chronicmyeloid leukemic cells were slightly, lessresponsive at all concentrations. Marrowcells from acute leukemic patients in remission exhibited a normal pattern ofresponsiveness. Average plasma CSF levels in leukemic patients were two to threetimes higher than the concentration of CSFin cultures maximally stimulated by monkey lung conditioned medium. The observed responsiveness of leukemic cells tostimulation by CSF-containing material isfurther evidence in support of the conclusion that most myeloid leukemias inman are conditioned, rather thanautonomous, neoplasms.

Submitted on July 23, 1973 Revised on November 1, 1973 Accepted on November 14, 1973     

Transcriptome Analysis of Human Glioblastoma Cells Susceptible to Infection with the Leningrad-16 Vaccine Strain of Measles Virus

Glioblastoma multiforme (GBM) accounts for almost half of all primary malignant brain tumors in adults and has a poor prognosis. Here we demonstrated the oncolytic potential of the L-16 vaccine strain of measles virus (MV) against primary human GBM cells and characterized the genetic patterns that determine the sensitivity of primary human GBM cells to oncolytic therapy. MV replicated in all GBM cells, and seven out of eight cell lines underwent complete or partial oncolysis. RNA-Seq analysis identified about 1200 differentially expressed genes (FDR < 0.05) with at least two-fold expression level change between MV-infected and uninfected cells. Among them, the most significant upregulation was observed for interferon response, apoptosis and cytokine signaling. One out of eight GBM cell lines was defective in type I interferon production and, thus, in the post-interferon response, other cells lacked expression of different cellular defense factors. Thus, none of the cell lines displayed induction of the total gene set necessary for effective inhibition of MV replication. In the resistant cells, we detected aberrant expression of metalloproteinase genes, particularly MMP3. Thus, such genes could be considered intriguing candidates for further study of factors responsible for cell sensitivity and resistance to L-16 MV infection.     

DNA Complementary to Viral RNA in Leukemic Cells Induced by Avian Myeloblastosis Virus

Nucleic acid hybridization studies were made between 71S-AMV-RNA and DNA from leukemic myeloblasts and from normal chicken cells. There was homology between the viral RNA and chicken cell DNA and to a greater extent between viral RNA and leukemic cell DNA. Leukemic cell DNA hybridized approximately twice as much viral RNA as did normal chicken DNA. Thermal melting studies showed that the viral RNA bound to normal and leukemic cell DNA consists of long polynucleotides (T(m) = 87 degrees and 92 degrees C, respectively, in 2x saline citrate). This suggests that the leukemic cells contain a DNA template of the viral RNA.     

Immunologic and Cytogenetic Studies of Chronic Lymphocytic Leukemic Cells

The lymphocyte transformation response of 17 chronic lymphocytic leukemiapatients when tested in the short-term tissue culture with PHA-M, and PPDwas found to be significantly decreased when compared to normal subjects.Serum factors were not found to be responsible for this cellular hyporesponsiveness. The proportions of immunoresponsive lymphocytes found in thepatients’ peripheral circulation decreased as their white blood cell count increased. The transformation response to PHA-M was generally better thanto PPD. Neither the PPD negative patients nor the normal PPD negativesubjects’ cells responded to PPD stimulation in vitro.

Monocytes usually would phagocytize particles added to the cultures andcould thus be distinguished from the nonphagocytic proliferating lymphocyteswhich were the only cells that incorporated thymidine H³. Radioautographsof tritiated thymidine also revealed the rate of PPD lymphocyte transformationto be slower than with PHA-M. There were no significant differences in theproportions or the degree of leukemic and normal transformed lymphocytelabeling with tritiated thymidine.

Cytogenetic studies revealed that the patients’ mitotic indices both in vivoand in vitro were markedly depressed. The modal chromosome number was46 in each patient, and no cytogenetic abnormalities other than those due toexposure to radiation were found.

Submitted on August 26, 1964 Accepted on November 22, 1964     

Colony Growth of Human Leukemic Peripheral Blood Cells In Vitro

The colony-forming potential of peripheral white blood cells from patientswith acute leukemia on normal humanperipheral WBC feeder layers has beenstudied. White blood cells from 12 of 20patients with acute granulocytic leukemiagave rise to large numbers of colonies,while WBC from eight patients withAGL, four patients with acute lymphocytic leukemia and three patients withacute stem cell leukemia gave rise to nocolonies or only small numbers. Coloniesformed from WBC of patients with AGLappear to go through a process of morphologic maturation to segmented granulocyte forms. Leukemic WBC will notserve as feeder layers in this system, butare not inhibitory in this respect. Thesignificance of these findings is discussed.

Submitted on February 19, 1971 Revised on May 3, 1971 Accepted on May 10, 1971     

Replication of Mouse-Tropic and Xenotropic Strains of Murine Leukemia Virus in Human × Mouse Hybrid Cells

The replication of mouse-tropic and xenotropic strains of murine leukemia virus in human x mouse hybrid cells was investigated. NB-tropic strains of the leukemia virus replicated efficiently in several hybrid lines, including those that contained a complete complement of human chromosomes and many mouse chromosomes. In lines with only a few mouse chromosomes, NB-tropic viruses failed to replicate. N- and B-tropic viruses replicated in human x N-type and human x B-type cells, respectively. The N- and B-tropic viruses replicating in these hybrid cells retained their original tropism. The viral restrictive functions of the mouse Fv-1 locus were expressed in the hybrid cells, restricting the replication of N- and B-tropic strains in human x B-type and human x N-type mouse cells, respectively. In contrast to mouse-tropic viruses, AT-124 virus, a xenotropic strain, replicated in human but not in mouse cells or in hybrid cells containing a complete complement of human chromosomes and near complete complement of mouse chromosomes However, hybrid lines with only a few mouse chromosomes supported AT-124 replication. Thus, human genes in hybrid cells do not restrict the replication of mouse or xenotropic murine leukemia virus strains, while mouse genes in such cells restrict xenotropic leukemia virus replication and, as determined by the mouse Fv-1 phenotype, mouse-tropic murine leukemia virus. These results indicate that exogenously applied mouse-tropic and xenotropic oncornaviruses exhibit different patterns of restriction in human-mouse hybrid cells and that such hybrid cells may be used for genetic analysis of oncornavirus replication.     

Virus-Specific Messenger RNA on Free and Membrane-Bound Polyribosomes from Cells Infected with Rauscher Leukemia Virus

Cells infected by Rauscher leukemia virus synthesize virus-specific RNA which can be detected by hybridization to the single-stranded DNA copy of the viral RNA. Evidence is provided that virus-specific RNA is present in free and membrane-bound polyribosomes of these cells. The relative content of virus-specific RNA, as measured by hybridization, is 6-10 times less on free polyribosomes than on membrane-bound polyribosomes. The messenger RNA associated with both classes of polyribosomes was characterized by density gradient centrifugation. In addition to a major RNA species identified as 36S RNA, at least 2 minor components in the 14S and 21S region have also been found. There is a striking difference in the distribution of these RNA species between free and membrane-bound polyribosomes.     

Chemical Induction of Focus-Forming Virus from Nonproducer Cells Transformed by Murine Sarcoma Virus

Focus-forming virus can be induced by chemicals from virus-negative clonal lines of cells transformed by murine sarcoma virus. The viruses activated from such nonproducer cells have the host range and serologic properties of endogenous helper viruses of the cells rather than those of the sarcoma virus used to transform them. The evidence indicates that murine sarcoma virus-transformed nonproducer cells of two species contain the genetic information for both murine sarcoma and helper virus production in an unexpressed form.     

Pyrimidine Metabolism in Man. II. Studies of Leukemic Cells

1. The activities of three enzymes involved in pyrimidine synthesis—aspartate carbamyltransferase, dihydro-orotase and dihydro-orotic dehydrogenase—were studied in sonicates of circulating leukocytes from 5 patients withmyelocytic leukemia, 2 with lymphocytic leukemia, 2 with myeloproliferativedisorders and 5 with infection. The erythrocytes from one patient with theDi Guglielmo syndrome were studied.

2. Neoplastic cells showed increased activities of all three enzymes tendingto parallel the cytologic evidence of immaturity. The increase of dihydroorotic dehydrogenase was the most striking abnormality. Leukocytes frompatients with infection or with myeloproliferative disorders showed similarbut much less marked alterations in the enzyme pattern.

3. Dihydro-orotic dehydrogenase, absent from mature erythrocytes, waspresent in the nucleated erythrocytes in the Di Guglielmo syndrome.

4. The enzyme 5 carboxymethylhydantoinase, previously found in somebacteria, was absent from normal and abnormal hemic cells.

Submitted on June 30, 1959 Accepted on September 27, 1959     

Relationship Between RNA-directed DNA Polymerase (Reverse Transcriptase) from Human Acute Leukemic Blood Cells and Primate Type-C Viruses

An RNA-directed DNA polymerase was isolated from the peripheral blood leukocytes of a patient with acute myelomonocytic leukemia by successive purification of a particulate cytoplasmic fraction with endogenous, ribonuclease-sensitive DNA polymerase activity. Like RNA-directed DNA polymerase from mammalian type-C virus, the human leukemic cell enzyme efficiently utilized (A)(n).(dT)(12-18) and (C)(n).(dG)(12-18) and had an approximate molecular weight of 70,000. Further, the leukemic cell enzyme was strongly inhibited by antisera to RNA-directed DNA polymerase of primate type-C virus in a fashion similar to that noted with an extensively purified RNA-directed DNA polymerase from a person with acute myelogenous leukemia [Todaro, G.J. & Gallo, R.C. (1973), Nature 244, 206]. By these biochemical and immunological results the leukemic cell enzyme could be differentiated from all other known cellular DNA polymerases but could not be distinguished from RNA-directed DNA polymerase of primate type-C virus. We interpret these data, combined with observations published elsewhere, to indicate that human acute myelogenous leukemia cells contain components related to primate type-C virus. The parameters used in this study may provide the specificity and sensitivity required for determining the presence or absence and (if present) the relatedness of RNA-directed DNA polymerase in other cases and types of human leukemia.     

Immunofluorescent Detection of Herpesvirus Antigens in Exfoliated Cells from Human Cervical Carcinoma

Exfoliated cells from patients with squamous carcinoma of the cervix contain antigens related to herpesvirus subtype 2, as revealed by direct or indirect immunofluorescent techniques. Normal squamous cells from the same subjects and from controls without the disease, and cells from a small number of tumors at sites other than the cervix, did not react with anti-herpesvirus subtype 2 serum. Antisera to adenovirus 18 or mycoplasma orale did not react with the exfoliated cells.     

Absence of a Specific Ganglioside Galactosyltransferase in Mouse Cells Transformed by Murine Sarcoma Virus

The ganglioside composition of a nonproducer subclone (derived from BALB/c 3T3 mouseembryo cells) transformed by Kirsten murine sarcoma virus was drastically altered compared to the nontransformed parental clone. The transformed clone was unable to synthesize the mono- and disialogangliosides, G(M1) and G(D1a), due to the complete absence of a specific galactosyltransferase. The lack of this enzyme activity was established by sensitive radiochemical and enzymological techniques in vivo and in vitro.     

人脐带血LAK细胞克隆化及其对白血病细胞的杀伤效应

重组人白细胞介素Ⅱ(rhlL-2)刺激下,脐带血单个核细胞在半固体培养中可增殖形成淋巴细胞克隆.大约20％的克隆细胞具有明显的LAK活性,对NK敏感的K562、NK抵抗的H7402及HL60、KG-1、Jurket(?)等多种白血病细胞均有强烈的杀伤作用,对HL60白血病祖细胞增殖(CFU-L)也有显著的抑制作用.     

Trans-Species Rescue of Defective Genomes of Murine Sarcoma Virus from Hamster Tumor Cells with Helper Feline Leukemia Virus

This report describes a trans-species rescue of defective MSV genome with helper leukemia virus derived from cats. This rescue was achieved by in vitro co-cultivation of hamster tumor cells with feline embryo cells in the presence of helper feline leukemia virus (FeLV), or by inoculation of tumor cells into FeLV-infected newborn cats.The rescued focus-forming viruses produced foci in feline embryo cultures but not in cultures of mouse, rat, and hamster species. One isolate was tested and found to induce sarcoma in a kitten. Antigenic and viral interference studies indicated that the focus-forming virus has the viral envelope of FeLV. Virus stocks consisted of a mixture of focus-forming particles and a 1000-fold excess of helper FeLV. Virus assay pattern in feline embryo cultures with or without added helper FeLV indicated that this helper virus is required for the transformation of feline cells.     

Helper Activity of Human Leukemic Tissue Extracts for Leukemia Virus Expression in Mice

Extracts of cultured human leukemic tissues increased the spleen focus-forming activity of Friend leukemia virus preparations in BALB/c and other partially resistant mice. Such mice carry the Fv-1(b) gene, which inhibits the expression of helper virus indigenous to the Friend virus complex, and allows co-infecting leukemia viruses of mice, cats, or chickens to substitute for the inhibited helper virus and increase the expression of the spleen focus-forming virus. The helper activity of extracts from human leukemic tissues shared several important properties with that associated with known leukemogenic viruses of animals.