Cytogenetic characterization of eight human lymphoblastoid cell lines.

Cytogenetic analyses of eight human lymphoblastoid cell lines that were established by a new procedure using B-cell growth factor (BCGF) and interleukin-4 (IL-4) revealed that three cell lines (37.5%) showed structural anomalies in more than 97% of their metaphases, whereas others were predominantly normal diploid. Our results indicate that a) the structural anomalies seen in three cell lines were not induced by culture technique but were constitutional defects present in donors' peripheral blood samples; b) one donor, whose blood gave rise to cell line BCDI, is a constitutional mosaic because both normal diploid and altered metaphases were present; and c) genetic instability should be monitored in regular blood donors because some of them may harbor blood-mediated clastogen or chromosome breakage factor(s) that could induce higher rates of recombinations in somatic cells of some recipients and thus may potentiate such individuals to develop certain types of malignancies.

A novel method of human B cell activation by liposomes coated with Fab' fragments of anti-IgM.

A novel method for activating fresh human B cell, using liposomes decorated with Fab' fragments of anti-human IgM antibodies (immunoloposomes), is described. In our immunoliposome preparation, more than 80% of Fab' were efficiently bound to liposomes and over 95% of the vesicles were uniformly coated with Fab'. Immunoliposomes could activate fresh B cells from peripheral blood of normal donors at least 100-fold better than soluble F(ab')2 fragments, as detected by [3H]uridine uptake. Such activation was also reflected in the enlarged cell size and highly increased [3H]thymidine uptake by B cells when human B cell growth factor (BCGF) was added. Density of Fab' on the liposomes surface did not affect the efficacy of immunoliposomes. The activation of B cells by immunoliposomes was short lived (less than 3 days), due perhaps to rapid internalization of the vesicles by B cells.

Isolation and characterization of growth factor(s) from a human B-cell lymphoma.

We demonstrate that human neoplastic B cells (Br cells) contain a cytoplasmic protein of molecular mass 60 Kd that exhibits B-cell growth factor (BCGF) activity on growth factor-dependent long-term human B cells as well as on autochthonous tumor cells. This 60-Kd protein is recognized by antibodies against a similar intracellular 60-Kd protein derived from normal human lymphocytes. These results demonstrate that the two proteins share epitope homology. Microculture bioassays indicate that neoplastic and normal 60-Kd proteins are capable of driving neoplastic B cells through S-phase. Western immunoblot analysis indicates that neoplastic B cells secrete 60- as well as 14-Kd protein. Immunoaffinity-purified proteins secreted by Br cells exhibit BCGF activity in anti-IgM or dextran sulfate-preactivated human B cells. In addition, a double-antibody immunofluorescence staining technique was used to demonstrate that Br cells express cell surface receptors for BCGF molecule(s). These studies provide support for the autocrine growth model for neoplastic human B cells and suggest that the autocrine growth factor derived from such tumor cells is similar if not identical to normal BCGF molecules.

Structural homology of interferon-gamma with B-cell growth factor and its proliferative effect on long term B-cell lines.

In this study, we have demonstrated that recombinant human interferon-gamma (IFN-gamma) acts as a B cell growth factor (BCGF) for long term B cell lines. Immuno dot blot analysis with polyclonal goat antibody against homogenously purified 60K intracellular BCGF (IC-BCGF) was shown to recognize IFN-gamma and IFN-alpha. Out of three E. coli derived recombinant IFN-gamma molecules tested, two containing the complete C-terminal were reactive with antibody and also exhibited BCGF activity, while the third lacking 15 amino acids from C-terminal did not exhibit BCGF activity nor any reaction to anti-IC-BCGF antibodies in immuno dot blot analysis. The proliferative effect of IFN-gamma on long term B cells could be blocked by anti-IC-BCGF antibodies in a dose dependent manner. These results indicated that this BCGF activity may be a result of structural homology between BCGF and C-terminal peptide of IFN-gamma.

Immunological evidence for the relation between low MR secreted form of human B cell growth factor and an intracellular 60K protein.

Homogeneously purified intracellular high-Mr BCGF (IC-BCGF) has been used as an immunogen for the development of heterotypic anti-IC-BCGF antibodies. Anti-IC-BCGF immunoglobulin has been purified by affinity chromatography and used for constructing an immunoaffinity matrix. This matrix was shown to recognize specifically IC-BCGF and the extracellular low-Mr secreted form of human BCGF (EC-BCGF). In the cytosolic extracts of lectin activated human PBLs two other proteins of 50K and 30K were also recognized by these antibodies. Immuno dot blot and western blot analysis also demonstrated that anti-IC-BCGF antibodies recognized EC-BCGF. These antibodies weakly interacted with human interferons (alpha, gamma). Immunoaffinity-purified IC-BCGF was shown to have a molecular weight of 60K, and it exhibited BCGF activity in long-term BCGF-dependent B cell lines. The Mr of one-step affinity-purified EC-BCGF was found to be 14K by SDS-PAGE analysis. This material demonstrated BCGF activity in both long-term BCGF-dependent B cell lines and anti-IgM-activated GoB cells, yet it did not exhibit IL-1 and IL-2 activities. We conclude that EC-BCGF is immunologically related to IC-BCGF through common structural epitopes.

Production of B cell growth factor(s) by neoplastic B cells from hairy cell leukemia patients.

Recent studies have shown that normal human T cells contain a high-molecular-weight (mol wt) protein exhibiting B cell growth factor (BCGF) activity. Other studies have shown that virally transformed human B cells also secrete a high-mol-wt BCGF-like molecule in vitro. We have studied neoplastic B cells from patients with untreated hairy cell leukemia (HCL) to ascertain whether such cytoplasmic BCGF activity is present in the tumor cells. Studies on HCL cells from four patients indicated that BCGF-like activity was in fact present in the cytosolic extracts when tested on autochthonous HCL cells as well as on normal BCGF-dependent human B cell lines. Chromatographic analysis indicated that the BCGF activity from HCL cells was similar in mol wt as well as function to the normal T cell-derived cytosolic BCGF activity. These studies suggest that HCL cells contain and, in some cases, secrete a high-mol-wt growth factor that can be autostimulatory and appears to resemble a similar growth factor molecule found in normal human T cells.

Purification and partial characterization of human intracellular B cell growth factor.

In the present report a protocol for the purification of an intracellular protein, exhibiting BCGF activity has been described. This protein is obtained from lectin stimulated human peripheral blood mononuclear cells and has been previously shown to be present only in the cytosolic extract of normal human T-lymphocytes. The isoelectric point (pI) of the purified protein is 6.3, as determined by both sucrose gradient isoelectric focusing and chromatofocusing. The molecular weight of the chromatofocused protein, determined by SDS-PAGE, is 60 kD. Thirty-three percent of the BCGF activity contained in the initial crude extract is recovered as a homogenously purified protein, with an estimated specific activity of 10(5) U/mg.

Partial purification and characterization of mitotic factors from HeLa cells.

Extracts from mitotic HeLa cells, when injected into Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) as evidenced by the breakdown of the germinal vesicle and the condensation of chromosomes. In this study we have attempted to purify and characterize these mitotic factors. When 0.2 M NaCl-soluble extracts of mitotic HeLa cells were concentrated by ultrafiltration and subjected to affinity chromatography on hydroxylapatite followed by DNA-cellulose, the proteins with MPA eluted as a single peak and their specific activity was increased approx. 200-fold compared with crude extracts. The molecular weight of the mitotic factors was estimated to be 100 kD as determined by chromatography on Sephacryl S-200. SDS-PAGE of the partially-purified mitotic factors indicated the presence of several polypeptides ranging from 40-150 kD with a major band of about 50 kD. The majority of these polypeptides were found to be phosphoproteins as revealed by 32P-labeling and autoradiography. Very little or no phosphorylation was observed at the 50 kD band. Several of these polypeptides were reactive with mitosis-specific monoclonal antibodies, MPM-1 or MPM-2, as shown by immunoblots of these proteins but the major polypeptide band at 50 kD was not. Removal of the immunoreactive polypeptides by precipitation with these antibodies did not destroy the MPA. The MPA of the crude or the partially-purified mitotic factors was destroyed by injection of (but not pretreatment with) alkaline phosphatase within 45 min after injection of mitotic factors. These results are discussed in terms of a possible role of phosphorylation-dephosphorylation of non-histone proteins in the regulation of mitosis and meiosis.

Growth factor-mediated proliferation in B cell non-Hodgkin's lymphomas.

The non-Hodgkin's lymphomas (NHLs) are a heterogeneous group of human lymphoid tumors, primarily of B cell lineage, which appear to represent arrested stages in B lymphocyte differentiation. Control of cell proliferation is a fundamentally important but poorly understood area of study in these tumors. We have studied a representative group of B cell NHLs to assess their potential for growth factor-mediated proliferation in vitro. Our results show that purified monoclonal NHL B cells of the small cell (well-differentiated lymphocytic lymphoma, nodular poorly differentiated lymphocytic lymphoma, etc) type, that were positive for the human malignancy-associated nucleolar antigen could be stimulated by human B cell growth factor (BCGF) to proliferate in vitro. Other B cell activators such as insoluble anti-Ig and the mitogen protein A also could stimulate thymidine incorporation in the lymphoma cell populations. In vitro lymphoma cell growth could be maintained in the presence of the growth factor for up to five weeks. The large B cell type NHL, however, appeared to be refractory to in vitro stimulation by BCGF as well as other stimulators of normal B cells. These studies suggest that human B cell lymphoid tumors are not only phenotypically similar to their normal B lymphocyte counterparts, but are also sensitive in some cases, to the same types of immunoregulatory molecules that control normal lymphoid cell growth.

Establishment of stable human T-T hybridomas.

Human T-T hybridomas potentially provide an invaluable resource for a variety of immunoregulatory molecules that modulate the immune response. To date, success in this technology, using human cell populations, has been hampered by several problems associated with proliferative and functional instability of the hybrid cells. These forms of instability are the result of a multifactorial process, with 1 parameter of importance being the chromosome number of the malignant parent cell line used for fusion. The present studies describe the production of a stable human T-T hybridoma generated by fusing a near diploid (modal chromosome number of 48) aminopterin-sensitive T cell line, CEM TG E11, and lectin-stimulated human peripheral blood lymphocytes. The rapidly growing hybrid cells have been clonally selected for the production of a B cell growth factor. Hybridization was documented by the presence of HLA phenotypes reflecting the combined antigens of the fusion partners. Fusions with 4 other partners besides CEM TG E11, where the majority of the cells had modal chromosome numbers ranging from 78 to 94, were proliferatively unstable. To date, hybrid cells derived from the CEM TG E11 fusion have been doubling approximately every 48 h for greater than 12 months, and selected clones constitutively produce B cell growth factor.

Intracellular human IL-1: a precursor for the secreted monokine.

An intracellular monocyte derived protein possessing interleukin 1 (IL-1) activity has been compared with the secreted from of IL-1. Our results indicated that this intracellular IL-1 had an apparent size greater than that of extracellular IL-1. Data are presented to show that a rabbit heterotypic antiserum prepared against epitopes on the secreted form of human IL-1 blocked the biologic activity of intracellular IL-1 in a dose dependent manner. Intracellular IL-1 had a mass of 30-40 kDa as determined by gel filtration using sephacryl-200 gel.

Evidence for an intracellular precursor for human B-cell growth factor.

Human B-cell growth factor has been described as a trypsin-sensitive protein of Mr 12,000-14,000. Evidence is provided herein that this relatively low molecular weight product may be released from a larger precursor molecule of Mr 60,000-80,000. The precursor protein is confined to the cytosol of freshly isolated T lymphocytes, and only the Mr 12,000-14,000 moiety is released upon lectin stimulation. The precursor protein was subjected to limited tryptic digestion, which demonstrated that the biologically active fraction of the moiety resided in a relatively low molecular weight fragment. The T lymphocyte routinely possessed an intracytoplasmic pool of the precursor protein, the amount of which cyclically varied depending upon its depletion by the secretion process of a lower molecular weight product. Analysis of the mRNA size coding for the majority of B-cell growth factor activity, determined by translation in Xenopus laevis oocytes, suggested that the B-cell growth factor-specific mRNA resided in the greater than or equal to 15S range. This value is consistent with the size of the larger precursor. Therefore, it is proposed that a precursor-product relationship exists for the processing of human B-cell growth factor, analogous to that which has been described for several other cytokines.

Phosphorylation of non-histone proteins associated with mitosis in HeLa cells.

Our previous studies indicated that certain non-histone proteins (NHP) extractable with 0.2 M NaCl from mitotic HeLa cells induce germinal vesicle breakdown and chromosome condensation in Xenopus laevis oocytes. Since the maturation-promoting activity of the mitotic proteins is stabilized by phosphatase inhibitors, we decided to examine whether phosphorylation of NHP plays a role in the condensation of chromosomes during mitosis. HeLa cells, synchronized in S phase, were labeled with 32P at the end of S phase, and the cells subsequently collected while they were in G2, mitosis, or G1. Cytoplasmic, nuclear, or chromosomal proteins were extracted and separated by gel electrophoresis. The labeled protein bands were detected by radioautography. The results indicated an 8-10-fold increase in the phosphorylation of NHP from mid-G2 to mitosis, followed by a similar-size decrease as the cells divided and entered G1. The NHP phosphorylation rate increased progressively during G2 traverse and reached a peak in mitosis. Radioautography of the separated NHP revealed eight prominent, extensively phosphorylated protein bands with molecular masses ranging from 27.5 to 100 kD. These NHP were rapidly dephosphorylated during M-G1 transition. Phosphorylation-dephosphorylation of NHP appeared to be a dynamic process, with the equilibrium shifting to phosphorylation during G2-M and dephosphorylation during M-G1 transitions. These results suggest that besides histone H1 phosphorylation, phosphorylation of this subset of NHP may also play a part in mitosis.

Association of an interleukin abnormality with the T cell defect in Hodgkin's disease.

The cellular immune defect in untreated Hodgkin's disease (HD) has long been recognized. This defect appears to be responsible for at least some of the morbidity and ultimately the mortality associated with the disease. In recent years, many studies have shown that the T cell component of the immune response is the apparent site where the defect in HD exists and where the immunoregulatory abnormalities that may account for the deficit are observed. The discovery of the lymphokines and monokines, comprising the human interleukin system, has elucidated some aspects of the regulatory control of the functional pathways involved in T lymphocyte activation and proliferation. The interleukin system can therefore provide the framework to dissect immunodeficiency states, such as that seen in HD. The present study indicates that HD patients' interleukin 1 (IL1) response appears to be normal, as is their T cell proliferative response to exogenous IL2. Interleukin 2 production by HD patients' peripheral blood mononuclear cells, however, is decreased when compared with age/sex-matched controls. The inability to generate IL2 after appropriate stimulation may reflect either a primary cellular defect or a regulatory defect, such as excessive immunosuppression, giving rise to the characteristic T cell hyporesponsiveness seen in HD.

Inactivation of mitotic factors by ultraviolet irradiation of HeLa cells in mitosis.

Extracts from mitotic HeLa cells, when injected into fully grown Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) indicated by germinal vesicle breakdown (GVBD) and chromosome condensation. Recently, we observed that the MPA of mitotic cell extracts is neutralized by the inhibitors of mitotic factors (IMF) in HeLa cells, which are activated at telophase and remain active throughout the G1 period. The activity of the IMF coincides with the process of chromosome decondensation, which begins at telophase and continues until the beginning of S phase, when chromatin reaches its most decondensed state. The objective of the present study was to investigate whether these two phenomena - chromosome decondensation and the activation of IMF - were related. The activity of IMF was measured in N2O-blocked mitotic HeLa cells, in which chromosome decondensation was induced by exposure to ultraviolet light, and subsequent incubation in medium containing inhibitors of DNA synthesis, hydroxyurea and arabinosylcytosine (araC). u.v. irradiation activated IMF was seen even at very high doses of X-irradiation. The IMF seemed to inactivate the mitotic factors directly by forming a complex that precipitated on heating at 60 degrees C for 15 min. Mg2+ or polyamines (i.e. spermine, spermidine, and putrescine), agents known to promote chromatin condensation partially restored the MPA of the u.v.-irradiated mitotic cell extracts. These results tend to support the conclusion that the IMF play a role in the decondensation of chromosomes.

Evidence for the presence of inhibitors of mitotic factors during G1 period in mammalian cells.

Our earlier studies indicated that the mitotic factors, which induce germinal vesicle breakdown and chromosome condensation when injected into fully grown Xenopus oocytes, are preferentially associated with metaphase chromosomes and that they bind to chromatin as soon as they are synthesized during the G2 phase. In this study, we attempted to determine the fate of these factors as the cell completes mitosis and enters G1. Extracts from HeLa cells at different points during G1, S, and G2 periods were mixed with mitotic extracts in various proportions, incubated, and then injected into Xenopus oocytes to determine their maturation-promoting activity. The maturation-promoting activity of the mitotic extracts was neutralized by extracts of G1 cells during all stages of G1 but not by those of late S and G2 phase cells. Extracts of quiescent (G0) human diploid fibroblasts exhibited very little inhibitory activity. However, UV irradiation of G0 cells, which is known to cause decondensation of chromatin, significantly enhanced the inhibitory activity of extracts of these cells. These factors are termed inhibitors of mitotic factors (IMF). They seem to be activated, rather than newly synthesized, as the cell enters telophase when chromosomes begin to decondense. The IMF are nondialyzable, nonhistone proteins with a molecular weight of greater than 12,000. Since mitotic factors are known to induce chromosome condensation, it is possible that IMF, which are antagonistic to mitotic factors, may serve the reverse function of the mitotic factors, i.e., regulation of chromosome decondensation.

Long-term growth of human B cells and their use in a microassay for B-cell growth factor.

Normal human B lymphocytes, prepared from peripheral venous blood, have been stimulated with intact anti-IgM (mu chain specific) bound to an insoluble matrix. The activation event, in a subfraction of human B cells, was associated with subsequent receptivity to the mitogenic effects of exogenously added B-cell growth factor. The ability of the cell population to specifically absorb the B-cell growth factor was dependent upon the time of stimulation with the anti-IgM. Continuous replenishment of the growth factor resulted in the ability to maintain long-term growth-factor-dependent human B-cell populations. These cultured B lymphocytes were shown to specifically absorb the B-cell growth factor, suggesting the presence of membrane receptors for it. The cultured B lymphocytes were routinely maintained in logarithmic-phase growth, in the presence of growth factor, with a population doubling time of 36 hr. These cultured B cells have been utilized in a microassay for the assessment of B-cell growth factor activity that is accurate, sensitive, and precise.

Chromosome-bound mitotic factors: release by endonucleases.

Additional evidence is presented to support our recently reported conclusion that the mitotic factors of mammalian cells, which induce germinal vesicle breakdown and chromosome condensation when injected into fully grown Xenopus laevis oocytes, are localized on metaphase chromosomes. Chromosomes isolated from mitotic HeLa cells were further purified on sucrose gradients and digested for varying periods with either the micrococcal nuclease or DNase II. At each time point of digestion the amount of mitotic factors released was determined by injecting a supernatant of these fractions, obtained by high-speed centrifugation, into oocytes. The amount of DNA rendered acid soluble under the conditions of digestion used was 3% ot 5% of the total chromosomal DNA. The extent of release of mitotic factors with both nucleases was estimated to be about 30% to 40% as evidenced by the reextraction of the undigested chromosomal pellet with 0.2 M NaC1. Similar results were obtained when nuclei from G2 cells were digested under identical conditions. The release of these chromosome-bound mitotic factors by mild digestion with these nucleases though only partial, clearly demonstrates that a significant proportion of these factors are localized on metaphase chromosomes.

Localization of mitotic factors on metaphase chromosomes.

The objective of this study was to determine whether the mitotic factors of HeLa cells, which induce meiotic maturation, i.e. germinal vesicle breakdown (GVBD) and chromosome condensation, when injected into fully grown Xenopus laevis oocytes, were localized in the cytoplasm or associated with the metaphase chromosomes. Cytoplasmic extracts were prepared by lysing mitotic HeLa cells in low-salt hypotonic buffer and separating the chromosomes by centrifugation. Th mitotic factors bound to chromosomes were extracted with high-salt (0.2 M-NaCl) buffer. Both the cytoplasmic and chromosomal protein fractions were evaluated for their maturation-promoting activity (MPA) in the Xenopus oocytes. The results of this study indicate that both the cytoplasmic and chromosomal fractions are identical in many respects, including their ability to induce GVBD, but the specific activity of the chromosomal fraction was at least threefold greater than that of the cytoplasmic fraction. These data suggest that a major portion of the mitotic factors is localized on the metaphase chromosomes. This association does not appear to be due to adventitious binding of mitotic proteins to chromosomes during the extraction procedures. Furthermore, when extracts were prepared in a similar way from early- and mid-G2-phase HeLa cells, only the nuclear extracts had MPA and no activity was found in the cytoplasmic fraction. Both the cytoplasmic and nuclear extracts of late-G2 cells exhibited MPA. These data support the conclusion that the mitotic factors become preferentially bound to chromatin as soon as they are synthesized, and as the cell synthesizes more of these factors in preparation for mitosis, increasing amounts of them are retained in the cytoplasm.