Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32 D.
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Animals↗
Biomedical subjects
G Rovera
Publications and source records attributed to G Rovera.
Point mutations in both transforming and non-transforming codons of the N-ras proto-oncogene of Ph+ leukemias.
The distribution and frequency of point mutations in the first and second coding exons of the N-ras proto-oncogene was examined in 6 cases of Philadelphia positive (Ph+) hemopoietic malignancies. To increase the detection sensitivity of the mutations and to estimate more accurately the frequency of abnormal alleles in the hemopoietic cell population, a polymerase chain reaction (PCR)/shotgun cloning/double stranded DNA sequencing method was used. Mutations activating the ras oncogenes involving codon 61 were observed in 5 out of 6 cases; in one of these cases (CML3), mutation at codon 61 involved a two base transition. Mutations involving codon 59 were also observed in one case (CML1). In longitudinal studies of 3 cases of chronic myelogenous leukemia samples obtained at the time of initial diagnosis and 5 to 7 years later, a multiplicity of mutations were detected at the time of initial diagnosis prior to any therapy. In one case (CML3), a mutation in codon 61 detected at diagnosis was still present 5 years later, in a second case (CML1) a mutation in codon 61 appeared during the course of the disease and persisted for at least one year, and in the third case (CML2) a mutation in codon 61 was present at diagnosis but absent 5 years later. In one instance (CML1) a mutation in codon 59 was present at the time of initial diagnosis but was not detectable in later samples. Several other point mutations leading to aminoacid changes were scattered predominately through the second exon but were not consistently detected in longitudinal studies on cells from the same patient. The data suggest that there is considerable genetic instability in the 2nd exon of N-ras in the myeloid leukemias but in every case a small subset of cells contains the mutations and these cells do not have a proliferative advantage.
Adult↗
Amplification of IL-2-driven T cell proliferation by recombinant human IL-3 and granulocyte-macrophage colony-stimulating factor.
Two recombinant human preparations of CSF, namely granulocyte-macrophage-CSF (GM-CSF) and IL-3 (multi-CSF), were tested for their ability to stimulate the growth of human freshly separated and in vitro activated lymphocytes. Both CSF independently induced short term proliferation in unfractionated PBL and lectin-stimulated T cells. Despite the great variability among different donors in the magnitude of lymphocyte response to the two growth factors, IL-3 at suboptimal concentrations (10 U/ml) consistently induced a higher proliferative response than did GM-CSF at suboptimal concentrations (5 ng/ml) in all of the preparations tested. When used in combination with IL-2, GM-CSF and, especially, IL-3 significantly potentiated the proliferative responses induced by IL-2 in both unstimulated and mitogen-activated lymphocytes. Dose-response curves using increasing concentrations of IL-2 and IL-3 and isobologram analysis of these interactions revealed a clear synergy of action between the two growth factors in inducing proliferation of unfractionated PBL, purified T cells, mitogen-activated lymphocytes, and alloantigen-stimulated T cells. In addition to enhancing the short term responsiveness to IL-2, GM-CSF and, especially, IL-3 drastically potentiated the long term growth of non-activated human lymphocytes and of lectin- or Ag-activated T cells in the presence of IL-2. Immunofluorescence analysis indicated a higher expression of activation Ag (anti-Tac receptors and HLA class II Ag) in cultures incubated in the presence of IL-3 either alone or in conjunction with IL-2. The overall data indicate that human GM-CSF and IL-3 can support the growth of cells within the lymphoid lineage and exert potent amplifying effects on IL-2-induced T cell growth in vitro.
Adjuvants, Immunologic↗
Expression of a distinctive BCR-ABL oncogene in Ph1-positive acute lymphocytic leukemia (ALL).
The Philadelphia chromosome (Ph1) is a translocation between chromosomes 9 and 22 that is found in chronic myelogenous leukemia (CML) and a subset of acute lymphocytic leukemia patients (ALL). In CML, this results in the expression of a chimeric 8.5-kilobase BCR-ABL transcript that encodes the P210BCR-ABL tyrosine kinase. The Ph1 chromosome in ALL expresses a distinct ABL-derived 7-kilobase messenger RNA that encodes the P185ALL-ABL protein. Since the expression of different oncogene products may play a role in the distinctive presentation of Ph1-positive ALL versus CML, it is necessary to understand the molecular basis for the expression of P185ALL-ABL. Both P210BCR-ABL and P185ALL-ABL are recognized by an antiserum directed to BCR determinants in the amino-terminal region of both proteins. Antisera to BCR determinants proximal to the BCR-ABL junction in CML immunoprecipitated P210BCR-ABL but not P185ALL-ABL. Nucleotide sequence analysis of complementary DNA clones made from RNA from the Ph1-positive ALL SUP-B15 cell line, and S1 nuclease protection analysis confirmed the presence of BCR-ABL chimeric transcripts in Ph1-positive ALL cells. In Ph1-positive ALL, ABL sequences were joined to BCR sequences approximately 1.5 kilobases 5' of the CML junction. P185ALL-ABL represents the product of a BCR-ABL fusion gene in Ph1-positive ALL that is distinct from the BCR-ABL fusion gene of CML.
Amino Acid Sequence↗
Signal transduction through the EGF receptor transfected in IL-3-dependent hematopoietic cells.
An expression vector for the epidermal growth factor (EGF) receptor was introduced into the 32D myeloid cell line, which is devoid of EGF receptors and absolutely dependent on interleukin-3 (IL-3) for its proliferation and survival. Expression of the EGF receptor conferred the ability to utilize EGF for transduction of a mitogenic signal. When the transfected cells were propagated in EGF, they exhibited a more mature myeloid phenotype than was observed under conditions of IL-3-directed growth. Moreover, exposure to EGF led to a rapid stimulation of phosphoinositide metabolism, while IL-3 had no detectable effect on phosphoinositide turnover either in control or EGF receptor-transfected 32D cells. Although the transfected cells exhibited high levels of functional EGF receptors, they remained nontumorigenic. In contrast, transfection of v-erbB, an amino-terminal truncated form of the EGF receptor with constitutive tyrosine kinase activity, not only abrogated the IL-3 growth factor requirement of 32D cells, but caused them to become tumorigenic in nude mice. These results show that a naïve hematopoietic cell expresses all of the intracellular components of the EGF-signaling pathway necessary to evoke a mitogenic response and sustain continuous proliferation.
Animals↗
Regulation of gene expression of myeloperoxidase during myeloid differentiation.
Myeloperoxidase (MPO) is a major heme enzyme involved in inflammatory responses of polymorphonuclear leukocytes. Using cDNA and intron specific probes for MPO we studied the regulation of MPO expression during myeloid differentiation of the promyelocytic HL-60 leukemia cell line. Mature MPO mRNA species of 3.3, 2.8 and 1.6 kb and heterogeneous nuclear (hn) RNA of greater than 8 and approximately 4 kb were observed in wildtype HL-60 cells. Induction of differentiation of the cells towards either granulocytes or macrophages resulted in a profound decrease (greater than 95%) in the concentration of MPO mRNA levels, showing that gene expression of MPO mRNA is closely linked to the stage of development of myeloid cells. Studies using normal and leukemic hematopoietic cells confirmed these findings and showed that myeloblasts and promyelocytes contain MPO mRNA. Rate of transcription of MPO was measured by a nuclear run-on assay in wild-type and day 3- and day -4 differentiated HL-60 cells and was nearly the same in all three. In contrast, rate of transcription of c-myc in the same nuclei became almost undetectable with induction of differentiation. Overall transcription decreased by 60% and 80% on day 3 and 4 of differentiation, respectively, compared to wild-type cells. Stability of mature MPO mRNA was also measured and found to be the same in wild-type and differentiated HL-60. Half-life of MPO hnRNA was less than or equal to 30 min in wild-type HL-60; nevertheless, this hnRNA was easily detectable 3 days after induction of differentiation of these cells. Taken together, the results show that decreased expression of MPO mRNA with differentiation occurs in part post-transcriptionally, possibly due to a failure in RNA processing. In addition, as overall transcription decreases during differentiation, MPO transcription is concomitantly reduced. This indicates that transcriptional and post-transcriptional mechanisms cooperate in the control of MPO gene expression.
Cell Differentiation↗
Myeloperoxidase and oncogene expression in GM-CSF induced bone marrow differentiation.
DNA synthesis, morphology, specific RNA accumulation and rates of specific protein synthesis in GM-CSF stimulated bone marrow progenitor cells were studied. DNA synthesis increased markedly for 64 hours and then gradually decreased to 5% maximal activity by 160 hours. Morphologic examination 40 to 64 hours after stimulation revealed an increasing proportion of immature myeloid cells. After this proliferative peak, cells differentiated into segmented neutrophils and monocytes/macrophages; only mature forms were present by 160 hours. Accumulation of mRNA for c-myb and c-myc was maximal at 40 hours just prior to maximal [3H]thymidine incorporation, while maximal accumulation of histone type 3 (H3) was coincident with maximal [3H]thymidine incorporation at 64 hours. As proliferation decreased and differentiation proceeded, levels of mRNA for c-myb and H3 decreased markedly, while levels of RNA for c-myc decreased gradually and remained elevated above day 0 levels. Levels of c-fos mRNA fluctuated slightly during the first 64 hours of culture and increased 13-fold by 160 hours when mature cells were present. Similarly, beta-2 microglobulin mRNA increased steadily to maximal levels at 112 to 160 hours which were 15-fold higher than day 0 levels. Myeloperoxidase (MPO) mRNA was present in maximal amounts at 40 to 64 hours after stimulation with GM-CSF as the number of immature myeloid cells peaked. Immunoprecipitation of MPO from pulse-labeled cell lysates demonstrated a 7-fold rise in synthetic rate of MPO of 64 hours and a 28-fold decline by 160 hours when only 5% immature myeloid cells were present. Thus, MPO protein synthesis closely follows MPO mRNA accumulation. Immunoprecipitation of lactoferrin, a marker of myeloid secondary granules, demonstrated a gradual 5-fold increase in synthetic rate as the cells matured. Taken together, these data show that maximal expression of the early myeloid differentiation enzyme myeloperoxidase in GM-CSF stimulated normal bone marrow cells occurs during peak proliferation of immature myeloid cells.
Animals↗
Recombinant human macrophage colony-stimulating factor (M-CSF) requires subliminal concentrations of granulocyte/macrophage (GM)-CSF for optimal stimulation of human macrophage colony formation in vitro.
Human macrophage colony-stimulating factor (M-CSF or CSF-1), either in purified or in recombinant form, is able to generate macrophagic colonies in a murine bone marrow colony assay, but only stimulates small macrophagic colonies of 40-50 cells in a human bone marrow colony assay. We report here that recombinant human granulocytic/macrophage colony stimulating factor (rhGM-CSF) at concentrations in the range of picograms enhances the responsiveness of bone marrow progenitors to M-CSF activity, resulting in an increased number of macrophagic colonies of up to 300 cells. Polyclonal antiserum against M-CSF did not alter colony formation of bone marrow progenitors incubated with GM-CSF at optimal concentration (1-10 ng/ml) for these in vitro assays. Thus, GM-CSF at higher concentrations (nanogram range) can by itself, elicit macrophagic colonies, and at lower concentrations (picogram range) acts to enhance the responsiveness of these progenitors to M-CSF.
Animals↗
Synergistic and antagonistic effects of recombinant human interleukin (IL) 3, IL-1 alpha, granulocyte and macrophage colony-stimulating factors (G-CSF and M-CSF) on the growth of GM-CSF-dependent leukemic cell lines.
Three human leukemia cell lines (TALL-101, AML-193, and MV4-11) that require granulocyte/macrophage-colony stimulating factor (GM-CSF) for growth in a chemically defined medium were examined for their response to recombinant human (rh) cytokines. Either rh interleukin (IL)-3 or rhGM-CSF alone supported the long term growth of all three cell lines, and the two growth factors acted synergistically to stimulate the proliferation of the early T lymphoblastic leukemia (TALL-101) and of the monocytic leukemia (AML-193) cells. However, IL-3 antagonized the proliferation of the biphenotypic B-myelomonocytic leukemia (MV4-11) cells in the presence of GM-CSF when both factors were used at very low concentrations. The rh granulocyte (G)-CSF independently supported the long and short term growth of AML-193 and MV4-11, respectively, and synergized with GM-CSF in inducing proliferation of these cells. By contrast, G-CSF did not stimulate TALL-101 cell growth and antagonized the effect of GM-CSF such that proliferation was arrested. Although neither rh macrophage (M)-CSF nor rhIL-1 alpha independently promoted proliferation of the three leukemia cell lines, these cytokines were able to either up- or down-regulate the GM-CSF-dependent growth of these cells. Taken together, these data demonstrate that leukemic cells often require the synergistic action of several cytokines for optimal growth, whereas other combinations of factors may be growth-inhibitory. This raises the possibility that multiple hemopoietic growth factors sustain or control leukemic cell proliferation also in vivo. In addition, the observation the G-CSF, M-CSF, and IL-1 alpha can, in some cases, arrest cell proliferation without inducing differentiation suggests that the programs of proliferative arrest and differentiation in leukemic cells can be dissociated.
Cell Division↗
Cytokine-dependent granulocytic differentiation. Regulation of proliferative and differentiative responses in a murine progenitor cell line.
Human granulocyte colony stimulating factor (G-CSF) can support the survival and short term proliferation of the interleukin 3 (IL 3)-dependent diploid murine hemopoietic progenitor cell line 32D C13. After 8 days in the presence of 30 U/ml of G-CSF and in the absence of IL 3, the great majority of 32D C13 cells becomes positive for myeloperoxidase (a marker that appears at the promyelocytic stage of the granulocytic lineage) and progressively differentiates into lactoferrin-containing neutrophilic granulocytes. Myeloperoxidase mRNA rapidly increases after 24 to 48 hr of treatment with G-CSF, peaks at day 6 and is no longer detectable at day 9 and 12, paralleling the appearance of myeloperoxidase-positive promyelocytes and myelocytes in the culture. After 12 days, 100% of the cells terminally differentiate, and clonogenic assays in IL 3-containing semisolid media indicate that the whole population has irreversibly lost proliferative capability. By using varying concentrations of both murine IL 3 and recombinant human G-CSF, the cultures develop an heterogeneous population of cells representing all the differentiation stages of the myeloid lineage, and the relative ratios of immature proliferating precursors and terminally differentiated cells present in the cultures can be modulated by modifying the concentrations of IL 3 or recombinant human G-CSF. Isobolic curves indicate that IL 3 and G-CSF have an antagonistic effect on the proliferation of 32D C13 cells. Thus, these cells represent a simplified in vitro model of normal granulocytic differentiation whose extent may be modulated completely in the presence of serum by two well-defined growth and differentiation factors: IL 3 and G-CSF.
Animals↗
Establishment and characterization of an undifferentiated human T leukemia cell line which requires granulocyte-macrophage colony stimulatory factor for growth.
A human leukemia cell line (TALL-101) was established from the bone marrow of a patient with an undifferentiated acute T cell leukemia using the conditioned medium (CM) of the human T cell leukemia virus (HTLV) II-transformed human cell line J-LB1. Immunofluorescence analysis on the original leukemic cells indicated the presence of T cell markers (Leu-1, Tdt, and T11); however, the established TALL-101 cell line expressed only antigens commonly present on progenitor cells, thymocytes, and myelomonocytic cells, but not on mature T cells. A high percentage of TALL-101 cells displayed the Tac antigen which was down-regulated upon incubation in the presence of recombinant human (rH) interleukin 2 (IL 2). Interferon (IFN)-gamma induced the appearance of class II histocompatibility leukocyte antigens (HLA) and of a T cell marker (3A1), and enhanced the expression of transferrin receptors on these cells. Further evidence for a T cell lineage of the TALL-101 cell line was provided by both chromosomic and genotypic analysis showing a translocation in chromosome 14 typical of T cell leukemias, and a rearrangement of the T-beta receptor locus. The growth-promoting activity in the J-LB1-CM was identified as granulocyte-macrophage colony stimulatory factor (GM-CSF), a growth factor which stimulates proliferation of normal myelomonocytic cells and other progenitor cells, but not known to have an effect on T cells. Dose response curves of [3H]thymidine incorporation and growth indicated that TALL-101 cells were sensitive to very low concentrations of rHGM-CSF, 5 ng/ml inducing maximal proliferation in chemically defined medium. The TALL-101 cell line is strictly GM-CSF-dependent for growth: upon depletion of GM-CSF from the culture medium, the cells stop proliferating immediately and die within 1 to 2 wk. The overall data, showing that GM-CSF is able to support the growth of a highly undifferentiated T cell leukemia, strongly suggests that this factor might have similar growth promoting effects on other immature T cell leukemias, and possibly, on normal T cell progenitors.
Antigens, Differentiation, T-Lymphocyte↗
Characterization of cDNA clones for human myeloperoxidase: predicted amino acid sequence and evidence for multiple mRNA species.
Myeloperoxidase is a component of the microbicidal network of polymorphonuclear leukocytes. The enzyme is a tetramer consisting of two heavy and two light subunits. A large proportion of humans demonstrate genetic deficiencies in the production of myeloperoxidase. As a first step in analyzing these deficiencies in more detail, we have isolated cDNA clones for myeloperoxidase from an expression library of the HL-60 human promyelocytic leukemia cell line. Two overlapping plasmids (pMP02 and pMP062) were identified as myeloperoxidase cDNA clones based on the detection with myeloperoxidase antiserum of 70 kDa protein expressed in pMP02-containing bacteria and a 75 kDa polypeptide produced by hybridization selection and translation using pMP062 and HL-60 RNA. Formal identification of the clones was made by matching the predicted amino acid sequences with the amino terminal sequences of the heavy and light subunits. Both subunits are encoded by one mRNA in the following order: pre-pro-sequences--light subunit--heavy subunit. The molecular weight of the predicted primary translation product is 83.7 kDa. Northern blots reveal two size classes of hybridizing RNAs (approximately 3.0-3.3 and 3.5-4.0 kilobases) whose expression is restricted to cells of the granulocytic lineage and parallels the changes in enzymatic activity observed during differentiation.
Amino Acid Sequence↗
Tumor-derived growth factors that support proliferation and differentiation of normal and leukemic hemopoietic cells.
The conditioned media of 34 human tumor cell lines were screened for the ability to induce granulocyte-macrophage colonies in vitro in bone marrow cultures, to stimulate proliferation of a murine IL-3 dependent hemopoietic cell line (32D clone 3) and to stimulate thymidine incorporation in suspension cultures of acute myelogenous leukemia cells. Twelve tumor cell lines produced factors that were active in these assays. The conditioned medium of the glioblastoma cell line U87 MG was characterized in detail and found to contain G-CSF and GM-CSF. Cloning and sequencing of the U87 MG G-CSF indicated that it was derived from G-CSF b mRNA, which encodes a protein with a deletion of 3 amino acids at residues 36-38. The gene for G-CSF was mapped to human chromosome 17 band q21, a region involved in translocations frequently found in acute promyelocytic leukemia. G-CSF (U87MG) was able to induce granulocytic differentiation of the total population of a murine IL-3 dependent cell line, 32D clone 3; this effect was antagonized by IL-3. GM-CSF (U87-MG) supported the proliferation without inducing differentiation of two growth factor-dependent leukemic cell lines, TALL 101 and AML-193.
Animals↗
Effect of Abelson murine leukemia virus on granulocytic differentiation and interleukin-3 dependence of a murine progenitor cell line.
The murine diploid hematopoietic cell line 32D Cl3 strictly requires interleukin-3 (IL-3) for proliferation. When 32D Cl3 cells are transferred to IL-3-free medium which contains recombinant human granulocyte colony stimulating factor (rhG-CSF), the cell number increases four- to five-fold, and after 14 days the whole cell population is differentiated into morphologically normal and myeloperoxidase- and lactoferrin-positive metamyelocytes and granulocytes. Infection with Abelson murine leukemia virus (A-MuLV) of 32D Cl3 cells growing in the presence of IL-3 induces, within 2 weeks, the appearance of cells that are IL-3-independent for growth. The latter cells lack myeloid, T and B cell markers, and are unable to differentiate, even in the presence of very high doses of rhG-CSF. However, once the 32D Cl3 cells have been exposed to G-CSF, they become resistant to the transforming effects of A-MuLV as judged by the appearance of the IL-3-independent clones. These findings suggest that the ability of Abelson virus to transform immature progenitor cells is due to interference of the v-abl gene product with the mechanisms that control the commitment of the cells to differentiate.
Abelson murine leukemia virus↗
Molecular cloning and characterization of a cDNA for human granulocyte colony-stimulating factor (G-CSF) from a glioblastoma multiforme cell line and localization of the G-CSF gene to chromosome band 17q21.
The conditioned media (CM) of the glioblastoma multiforme cell line, U87 MG, contains abundant granulocyte colony-stimulating factor (G-CSF) activity (Tweardy et al., 1987). An oligonucleotide encoding the amino acids -11 to -4 of G-CSF detected a single abundant G-CSF mRNA of 1.6 kilobases (Kb) produced by U87 MG cells. Screening of a U87 MG cDNA library with the oligonucleotide identified cDNA clones of 1.3-1.4 Kb. Sequencing of one clone (pG-CSF6) confirmed that it encoded G-CSF and was derived from G-CSFb mRNA encoding a protein with a three amino acid deletion at positions 36-38. Only a single base substitution was observed at the third position of the codon for leu 152 indicating that G-CSF is highly conserved in cells of widely different origin. Somatic cell hybridization studies and chromosomal in situ hybridization localized the G-CSF gene to the long arm of chromosome 17 in band 17q21, proximal to the 17q breakpoint characteristic of acute promyelocytic leukemia.
Amino Acid Sequence↗
Localization of myeloperoxidase to the long arm of human chromosome 17: relationship to the 15; 17 translocation of acute promyelocytic leukemia.
Myeloperoxidase (MPO) is an enzyme whose synthesis is restricted to the promyelocytic stage of myeloid differentiation. We have recently described the cloning and sequencing of a cDNA for MPO. Using a regional mapping panel of somatic cell hybrids containing various deleted or translocated segments of chromosome 17, we have assigned MPO to a region between 17q21 and 17q23. In situ hybridization refined this localization in that grains on chromosome 17 were significantly clustered at bands q22-23 and no hybridization was detected at q21. In light of this chromosome assignment, the relationship of MPO to the 17q translocation breakpoint characteristic of acute promyelocytic leukemia (APL) was considered. Because this breakpoint has been variously assigned to different bands on 17q from 17q11.2 to 17q22, the cytogenetic and molecular distance between this breakpoint and MPO cannot be accurately determined. MPO and other probes mapped to this region of 17 will be important in searching for altered Southern blot patterns after conventional or pulsed-field gel analysis of DNA from APL patients.
Chromosome Mapping↗
Characterization of the human PIM-1 gene: a putative proto-oncogene coding for a tissue specific member of the protein kinase family.
The mouse PIM-1 gene is involved in the pathogenesis of virally-induced mouse lymphomas. We have cloned and analyzed the human homologue of the mouse PIM-1 gene to investigate its role in human lymphoma and leukemia. Overlapping cDNA clones from a K562 (human erythroleukemia cell line) library were isolated and sequenced. The deduced amino acid sequence showed significant homology to a number of the protein kinases but did not have a transmembrane region. Genomic clones from the 380 cell line (human B cell leukemia) were analyzed. The PIM-1 transcript was found to derive from 5 Kb of genomic DNA. Six exons and five introns were identified. The promoter region has no TATA or CAAT boxes, but did have multiple potential Sp1 binding sites (CCGCCC). Studies of expression of this gene using Northern blots of human cell lines showed it to be transcribed primarily in B lymphoid and myeloid cell lines. The characterization of the human PIM-1 gene will allow the definition of its role in hemopoietic malignancies and in hematolymphoid differentiation.
Amino Acid Sequence↗
A suppressor lymphokine produced by human T leukemia cell lines. Partial characterization and spectrum of activity against normal and malignant hemopoietic cells.
Human T leukemia cell lines spontaneously release into their medium a suppressor lymphokine, T leukemia-derived suppressor lymphokine (TLSL), able to inhibit proliferation, DNA synthesis, and colony formation in a variety of malignant hemopoietic cell lines, as well as in normal myelomonocytic progenitor cells from bone marrow and peripheral blood. Titration curves indicated that the inhibitory activity in the crude supernatant preparations ranged from 10(-3)-10(-9): the supernatants from CCRF/CEM, HUT-78, and MOLT-4 cell lines were the most active, those from HPB-ALL, JM, and CCRF/HSB2 displayed an intermediate activity, and the Jurkat supernatant was the least active. Target cell lines of B cell origin (Burkitt lymphomas) were more sensitive than granulocytic, monocytic, erythroid, and T cell lines. Partial purification by ammonium sulfate precipitation and column chromatography demonstrated that TLSL is a protein with an Mr of 88,000, as determined by gel filtration. A high Mr form (greater than 300,000) was produced in serum-free medium by one of the most active producer cell lines (CCRF/CEM), and appeared to be an aggregate of the 88,000 Mr form. Neither the partially purified fractions obtained nor the crude supernatant preparations displayed antiviral activity or contained interleukin 2. Unlike lymphotoxin and tumor necrosis factor, TLSL is cytostatic: maximal inhibition of proliferation was observed 4-5 d after addition of crude supernatant to the target cells, and was not accompanied by a significant loss in cell viability. The antiproliferative capacity of TLSL was manifested both in suspension and methylcellulose cultures. Treated target cells accumulated either in the G1 or in the S phase of the cell cycle. The effect of TLSL on the target cells is irreversible: even brief (1 h) incubation of sensitive cells with TLSL resulted in inhibition of proliferation measured 5 d later. Although TLSL is produced by leukemic T cell lines, this lymphokine inhibits proliferation of normal peripheral blood T cells in response to mitogens or alloantigens: T lymphocyte activation was inhibited by all of the T cell supernatants tested. In contrast, when T cell lines were used as targets, no inhibition of proliferation was detected with two exceptions: the low producer Jurkat cell line was sensitive to all the T cell-derived supernatants, and the intermediate producer CCRF/HSB2 cell line was sensitive only to the three most active supernatants, CCRF/CEM, MOLT-4, and HUT-78. The possible significance of TLSL and its relationship with other suppressor lymphokines previously described in other systems is discussed.
Cell Cycle↗