Characterization of the reverse transcriptase from a new retrovirus (HTLV) produced by a human cutaneous T-cell lymphoma cell line.
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Cell Line↗
Biomedical subjects
F W Ruscetti
Publications and source records attributed to F W Ruscetti.
Regulation of human T-cell proliferation: T-cell growth factor and isolation of a new class of type-C retroviruses from human T-cells.
The discovery, characterization, and purification of human T-cell growth factor (TCGF) has led to the establishment of continuously growing T-lymphoblast cell lines from normal people and from patients with certain T-cell neoplasias. In contrast to normal T-cells, neoplastic mature T-cells respond directly to TCGF, requiring no prior lectin or antigen in vitro activation. The transformed T-cell lines have phenotypic characteristics consistent with the neoplastic cells of their disease of origin. A novel retrovirus, human T-cell lymphoma-leukemia virus (HTLV), has been isolated from the fresh and cultured cells of two of these patients. Subsequent characterization of this virus has shown that it is not significantly related to any known animal retrovirus, is not an endogenous (genetically transmitted) virus of man, and so far has been associated only with fresh or cultured T-cells from patients with T-cell neoplasia. These results suggest that HTLV infected some mature T-cells of some people and that it might be involved in some neoplasias involving these cells.
Adult↗
T-cell growth factor and the culture of cloned functional T cells.
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Animals↗
Characterization and distribution of nucleic acid sequences of a novel type C retrovirus isolated from neoplastic human T lymphocytes.
A type C retrovirus (designated HTLV) recently isolated from a cell line derived from a lymph node and later from peripheral blood of a person with cutaneous T-cell lymphoma (mycosis fungoides) was characterized by nucleic acid hybridization experiments. HTLV [3H]cDNA hybridized 90% to its own 70S RNA with kinetics consistent with the genetic complexity of other retroviruses, but it did not hybridize substantially to RNA or proviral DNA from any animal retroviruses (types B, C, and D), including those from nonhuman primates. Conversely, [3H]cDNA from other retroviruses did not hybridize to RNA or DNA of the human T-cell line producing HTLV. HTLV proviral sequences were present (two to three copies per haploid genome) in DNA of these cells, and homologous sequences were present in the cell cytoplasmic RNA (0.3% viral sequences by weight). HTLV-related nucleic acid sequences were not found in DNA from various other human tissues. The results indicate that HTLV is a new class of type C virus that is not an endogenous (genetically transmitted) retrovirus in man.
Base Sequence↗
Immunological properties of a type C retrovirus isolated from cultured human T-lymphoma cells and comparison to other mammalian retroviruses.
HTLV strain CR (HTLVCR) is a retrovirus which was isolated from a human T-cell lymphoma cell line. A protein of molecular weight 24,000 p24, was purified from this virus. Several results indicate that this p24 is an internal core protein of HTLVCR. (i) The p24 copurified with viral cores. (ii) It was labeled with 125I after disruption of the virus, but not when undisrupted virus was iodinated. (iii) The amount of p24 was directly proportional to the amount of HTLVCR. (iv) In chromatographic properties, the HTLVCR p24 behaved similarly to the major structural protein (24,000- to 30,000-molecular-weight protein) of other retroviruses. A rabbit antiserum raised against disrupted HTLVCR precipitated the labeled p24, and the precipitation was competed for by unlabeled HTLVCR and by cytoplasmic proteins from cells producing HTLVCR, but not by proteins from normal human cells, including normal growing human T-cells, and several cultured human cutaneous T-cell lymphoma lines. Proteins from several mammalian type B, type C, and type D viruses also failed to compete in this precipitation. Moreover, HTLVCR did not react in homologous and interspecies assays for p30 antigens of several mammalian type C and type D viruses. These observations agree with immunological comparisons between reverse transcriptase of HTLVCR and other retroviruses and nucleic acid sequence homology studies which indicate that the various HTLVCR isolates represent new retroviruses found in some human T-cell neoplasias.
Antigens, Viral↗
Effect of fibroblast, lymphoid, and myeloid interferons on human tumor colony formation in vitro.
A variety of solid and hematological human tumors and normal human bone marrow specimens were assayed for colony formation in a short-term soft-agar culture system. The effect of human fibroblast, lymphoid, and myeloid interferons on inhibition of colony formation was assessed. The effect of interferon on colony formation formed a continuum from complete inhibition to stimulation of growth. Of 40 evaluable tumor specimens, 18 showed at least a 70% inhibition of colony formation in the presence of interferon, at concentrations of 1000 units/ml or less. Four specimens (acute myelogenous leukemia, osteogenic sarcoma, neuroblastoma, ovarian carcinoma) showed at least 3-fold stimulation of colony formation with interferon. Two of 12 normal bone marrow specimens grown with colony-stimulating, factor-conditioned media showed greater than 70% colony inhibition with interferon. A dose-response relationship was seen in all tumor specimens tested. While fibroblast interferon was the most active in this system, all interferons showed the same magnitude and direction of activity. Continuous exposure and 1-hr incubation of tumor cells with interferon were identical in terms of colony inhibition. These data support the ability of this assay system to select tumors responsive in vitro to interferon, suggest the optimal species and concentration for inhibition or stimulation of growth, and support a direct role of interferon in the regulation of cell growth independent of other immunoregulatory actions of interferon. Such information may prove useful for predicting response in vivo to interferon in Phase II trials.
Bone Marrow Cells↗
Restricted expression of retrovirus nucleic acids and proteins in primate type C virus (gibbon ape leukemia virus-simian sarcoma virus)-initiated human B-lymphoblast cultures.
Fresh human B-lymphoblasts established in culture following exposure of adult peripheral blood leukocytes to type C retroviruses of the simian sarcoma virus/simian sarcoma-associated virus-gibbon ape leukemia virus group were analyzed in detail for the presence of the infecting virus. Viral expression ranged from production of low levels of intact virus in a few cultures to the presence of viral RNA and protein in the absence of detectable of levels of complete virus in the majority of the cultures. In situ molecular hybridization assays using 3H-labeled complementary DNA and indirect immunofluorescence assays using antibody to purified viral protein indicated that the expression of viral RNA and proteins are preferentially expressed in only a fraction of the cells in some cultures. If expression of the infecting viral sequences is necessary for the sustained growth of these cells, then those cells detectably synthesizing viral RNA and proteins may be influencing the growth of the remaining virus-negative cells. The lack of virus production in cultures synthesizing viral RNA and protein indicate that these human B-lymphocytes restrict the life cycle of these viruses at some step(s) after transcription of viral RNA or translation of viral protein.
B-Lymphocytes↗
Long-term suspension cultures of human cord blood myeloid cells.
A method is described that allows for the routine long-term (greater than 3 mo) growth of normal, immature human myeloid cells in liquid suspension culture. The techniques employ cell separation, utilization of special growth conditions (RPMI with hydrocortisone and vitamin D), and cord blood as the source of leukocytes. These cultures are composed predominantly of immature myeloid cells that have grown for over 3 mo in culture but eventually terminate as differentiated, mature granulocytes and monocyte-macrophages. Application of these techniques to other sources of fresh human leukocytes (bone marrow and adult peripheral blood) resulted in only short-term proliferation of myeloid cells. The techniques described can be used for the routine expansion of immature myeloid and monocytoid cell populations, particularly from newborns, and should be useful for systematic studies of normal myeloid-monocytoid cell growth and differentiation and providing normal control cells in studies employing human leukemic myeloid cells.
Bone Marrow Cells↗
Clonal analysis of the response of human myeloid leukemic cell lines to colony-stimulating activity.
The recent development of two continuously proliferating human myeloid leukemic cell lines (HL-60 and KG-1) that response to CSA provides an opportunity for a detailed study of the interaction of CSA with leukemic myeloid cells. Here we report on the colony-forming ability of HL-60 and KG-1 over an extended culture life of the cells. Several different sources of human CSA of different stages of purity enhanced colony formation of these cells. CSA, obtained from conditioned media from an SV-40 transformed human trophoblast, was partially purified, and its activity for normal bone marrow copurified with the activity that stimulated HL-60 colony formation. Over 100 clones of HL-60 were developed and tested for their response to CSA. All responded to CSA by showing an increase in colony size and number. However, none of the colonies formed from any of the 100 clones differentiated in response to CSA despite the fact that many chemical can induce differentiation of HL-60. since HL-60 forms spontaneous colonies without the addition of any exogenous stimulating factors, HL-60 conditioned media and cell extracts were tested for the production by these cells of their own endogenous growth-promoting activity (such as a CSA-like molecule). No growth-promoting endogenous activity was found that stimulated normal bone marrow or HL-60 colony formation even after concentration and fractionation methods were employed. These experiments suggest that: (1) the effect of CSA markedly favors proliferation over differentiation in these cell lines; (2) CSA is unlikely to suppress growth of the age of the type of leukemic myeloid cells that HL-60 and KG-1 represent; and (3) if HL-60 cells produce their own growth-promoting factor it is not detectable in the media.
Cell Line↗
Spontaneous release of a factor with properties of T cell growth factor from a continuous line of primate tumor T cells.
A continuous lymphoid cell line had been previously established from a gibbon with spontaneous lymphosarcoma. This cell line, designated as MLA144, when tested after several years in culture was shown to release spontaneously a factor biologically and biochemically similar to human T cell growth factor (TCGF). Conditioned media (CM) from MLA144 cells support growth and DNA synthesis of T cells from humans, several other species of primates, and also from mice and rabbits. The activity in the MLA144 CM is resistant to 60 degrees C and to low and high pH, has a m.w., as determined by gel filtration, of 21,500, elutes from DEAE-cellulose at 0.04 to 0.06 M sodium phosphate buffer, pH 7.6, and has an isoelectric point of about 6.45. Surface-marker analysis of MLA144 cells by rosetting techniques indicates that they are T cells lacking in the receptor for the Fc portion of IgG. The release of TCGF by MLA144 cells should have practical value in terms of ease of TCGF production and should be of great help in the facilitation of studies on the cell biology and molecular biology of TCGF production.
Animals↗
Human T-lymphocyte growth factor: regulation of growth and function of T lymphocytes.
The discovery of T-cell growth factor (TCGF) has made it possible to now routinely grow in tissue culture normal and neoplastic human T cells for long periods and in large amounts. TCGF has been recently purified. It is a small protein released by a subset of mature T cells following lectin-antigen activation, which in turn acts upon other T-cell subsets that have developed specific receptors for TCGF after lectin-antigen stimulation. Thus, release of TCGF and development of receptors for it appear to be obligatory for the clonal expansion of all activated T cells. Unlike normal T cells, neoplastic T cells respond directly to TCGF, requiring no prior in vitro lectin-antigen activation. This has led to the development of several new cell lines from patients with T-cell leukemias and lymphomas. In some cases, these cells become independent of exogenous TCGF by producing their own growth factor, implying a role for TCGF in the continuous proliferation of these cells. These developments necessitate a reevaluation of some concepts of immunoregulation of T-cell activities in terms of production and response to TCGF. In addition, this information has clinical implications. Recent results have shown that a major defect of the athymic nude mouse is the inability to produce TCGF and that some immunosuppressive agents, such as glucocorticosteroids and cyclosporin-A, exert their effects on T cells by disrupting the TCGF-T-cell interaction. Some human immune deficiencies might be due to a failure to respond to or to produce TCGF, which in some cases might be corrected by exogenous TCGF.
Animals↗
Human T-cell growth factor: parameters for production.
Using conditioned media (CM) from phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBL) we observed long-term selective growth of T-cells from normal human donors. This T-cell growth was continuously dependent on addition of a factor called T-cell growth factor (TCGF). The optimal method for preparing highly active CM from single donor PBL involves the addition of mitomycin C-treated B-lymphoblastoid cell lines to the mixture of PBL and PHA. A number of different cell lines greatly augmented the production of TCGF in 18/18 cases. Preparation of plasma membranes from the Daudi cell line could replace the intact cells in the production of TCGF but those from the cell line, Molt-4, could not. Since the cell surface of Daudi possesses HLA-D antigens but not HLA-A, B, and C, and Molt-4 has HLA-A and B and not HLA-D, it is possible that the Ia antigens (HLA-DRw in man) are important in the release of TCGF. Using this method for growth factor production, an analysis was made concerning the events necessary for lymphocyte activation and the requirements for production and release of TCGF. Removal of PHA 12 h after incubation had no effect on lymphocyte transformation but decreased TCGF release by 90%. In addition, colchicine and cytosine arabinoside inhibited DNA synthesis but had no effect on TCGF release. Little or no TCGF activity was present after cellular protein synthesis was inhibited by puromycin and cycloheximide. These results suggest that TCGF production: a) requires protein synthesis; b) requires binding of the stimulating agent; c) can occur in a non-dividing cell, probably a terminally differentiated T-cell, without the need for cellular proliferation; and d) needs the assistance of an adherent cell which probably is a monocyte-macrophage. The ability to produce TCGF from single human donors will allow better understanding of the nature and action of TCGF.
Cell Division↗
Functional and molecular characteristics of T-cell growth factor.
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Animals↗
T-cell lines established from human T-lymphocytic neoplasias by direct response to T-cell growth factor.
Long-term growth of lymphoblastoid T cells from tissue samples from six of six patients with cutaneous T-cell lymphoma (CTCL) and six of six patients with acute T-lymphoblastic leukemia (ALL) has been achieved by using partially purified mitogen-free human T-cell growth factor (pp-TCGF). One cell line, CTCL-2, is now independent of added growth factor; the others continue to show absolute dependency on its presence. All lines have been in continuous culture for at least 4 months and some for > 1 year. They are erythrocyte-rosette positive and are negative for Epstein-Barr virus nuclear antigen. Most of the lines are negative for Fc and complement receptors and for surface immunoglobulin except that CTCL-1 and CTCL-2 have some cells positive for these cell surface markers. Results of histochemical studies on these cell lines are similar to the known patterns for fresh cells from their disease of origin. Cell line CTCL-3 has an abnormal karyotype, but no detectable chromosomal abnormalities were found in the other lines, consistent with the karyologic features of their clinical sources. Because T cells from normal donors do not respond to pp-TCGF unless the cells are first "activated" by a lectin mitogen such as phytohemagglutinin or an antigen, the direct response to pp-TCGF of T cells from patients with T-cell neoplasias suggests that the cell lines represent a transformed neoplastic cell population. Although some of the cell lines may be normal T cells activated by the malignant cells, the morphologic and histochemical properties of the cell lines, the abnormal karyotype of CTCL-3, and the independent growth of CTCL-2 support the conclusion that most of these cell lines are of malignant origin.
Antigens, Surface↗
Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma.
Retrovirus particles with type C morphology were found in two T-cell lymphoblastoid cell lines, HUT 102 and CTCL-3, and in fresh peripheral blood lymphocytes obtained from a patient with a cutaneous T-cell lymphoma (mycosis fungoides). The cell lines continuously produce these viruses, which are collectively referred to as HTLV, strain CR(HTLV(CR)). Originally, the production of virus from HUT 102 cells required induction with 5-iodo-2'-deoxyuridine, but the cell line became a constitutive producer of virus at its 56th passage. Cell line CTCL-3 has been a constitutive producer of virus from its second passage in culture. Both mature and immature extracellular virus particles were seen in thin-section electron micrographs of fixed, pelleted cellular material; on occasion, typical type C budding virus particles were seen. No form of intracellular virus particle has been seen. Mature particles were 100-110 nm in diameter, consisted of an electron-dense core surrounded by an outer membrane separated by an electron-lucent region, banded at a density of 1.16 g/ml on a continuous 25-65% sucrose gradient, and contained 70S RNA and a DNA polymerase activity typical of viral reverse transcriptase (RT; RNA-dependent DNA nucleotidyltransferase). Under certain conditions of assay, HTLV(CR) RT showed cation preference for Mg(2+) over Mn(2+), distinct from the characteristics of cellular DNA polymerases purified from human lymphocytes and the RT from most type C viruses. Antibodies to cellular DNA polymerase gamma and anti-bodies against RT purified from several animal retroviruses failed to detectably interact with HTLV(CR) RT under conditions that were positive for the respective homologous DNA polymerase, demonstrating a lack of close relationship of HTLV(CR) RT to cellular DNA polymerases gamma or RT of these viruses. Six major proteins, with sizes of approximately 10,000, 13,000, 19,000, 24,000, 42,000, and 52,000 daltons, were apparent when doubly banded, disrupted HTLV(CR) particles were chromatographed on a NaDodSO(4)/polyacrylamide gel. The number of these particle-associated proteins is consistent with the expected proteins of a retrovirus, but the sizes of some are distinct from those of most known retroviruses of the primate subgroups.
Adult↗
Normal functional characteristics of cultured human promyelocytic leukemia cells (HL-60) after induction of differentiation by dimethylsulfoxide.
The HL-60 human promyelocytic leukemia cell line can be induced to terminally differentiate to mature myeloid cells sharing a number of functional characteristics with normal granulocytes including response to chemoattractants, development of complement receptors, phagocytosis, superoxide production, and nitroblue tetrazolium dye reduction. Hence the Me2SO-induced HL-60 cells provide a unique in vitro model for studying various important aspects of human myeloid cell differentiation.
Binding Sites↗
T-cell growth factor-mediated T-cell proliferation.
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Absorption↗
Detection of HLA-DRw (Ia-like) antigens on human T lymphocytes grown in tissue culture.
Human T lymphocytes that proliferate in the presence of conditioned medium from PHA-stimulated allogeneic peripheral blood cells were shown to express IPA antigens after the 8th culture day. Ia antigens as detected by xenogeneic antisera were present on 80 to 90% of the cultured cells which were also strongly reactive with xenogeneic antisera defining a human T cell antigen and formed E rosettes. Control cultures with PHA or no conditioned medium expressed T cell but not Ia antigens. These cultured T cells also express the same HLA-DRw determinants as the B cells of the donor they were derived from. Absorption of xenogeneic Ia, and HLA-DRw alloantisera with cultured T cells completely removed the reactivity of these sera for enriched peripheral blood B lymphocytes from normal donors.
Animals↗