Membrane alterations associated with “transformation” by BUdR in BUdR-dependent cells : Fluorescence polarization studies with a lipid probe

Steady state and nanosecond fluorescence polarization studies were carried out on membranes of a “bromodeoxyuridine (BUdR) dependent” cell line (B4) derived from a malignant Syrian hamster melanoma line. When grown in the presence of BUdR B4 cells resemble transformed cells (in terms of several biological characteristics), while B4 cells grown in the absence of BUdR resemble untransformed cells. B4 cells were labelled with the lipid probe 1,6-diphenyl-1,3,5-hexatriene, which had been used previously to show that fluorescence polarization values of membrane lipids of virally transformed cells are higher than fluorescence polarization values of membrane lipids of untransformed cells. The steady state fluorescence polarization values of membrane lipids of B4 cells in BUdR were found to be larger than those of cells in the absence of BUdR, and the change in fluorescence polarization values was found to be fully reversible. Nsec rotational correlation time experiments confirmed and extended the steady state results. The results of the fluorescence polarization studies suggest that the membranes of B4 cells grown in the presence of BUdR resemble those of virally transformed cells while membranes of B4 cells grown in the absence of BUdR resemble those of untransformed cells.     

Expression of constitutively active Akt/protein kinase B signals GLUT4 translocation in the absence of an intact actin cytoskeleton

The actin cytoskeleton has been shown to be required for insulin-dependent GLUT4 translocation; however, the role that the actin network plays is unknown. Actin may play a role in formation of an active signaling complex, or actin may be required for movement of vesicles to the plasma membrane surface. To distinguish between these possibilities, we examined the ability of myr-Akt, a constitutively active form of Akt that signals GLUT4 translocation to the plasma membrane in the absence of insulin, to signal translocation of an HA-GLUT4-GFP reporter protein in the presence or absence of an intact cytoskeleton in 3T3-L1 adipocytes. Expression of myr-Akt signaled the redistribution of the GLUT4 reporter protein to the cell surface in the absence or presence of 10 microm latrunculin B, a concentration sufficient to completely inhibit insulin-dependent redistribution of the GLUT4 reporter to the cell surface. These data suggest that the actin network plays a primary role in organization of the insulin-signaling complex. To further support this conclusion, we measured the activation of known signaling proteins using a saturating concentration of insulin in cells pretreated without or with 10 microm latrunculin B. We found that latrunculin treatment did not affect insulin-dependent tyrosine phosphorylation of the insulin receptor beta-subunit and IRS-1 but completely inhibited activation of Akt/PKB enzymatic activity. Phosphorylation of Akt/PKB at Ser-473 and Thr-308 was inhibited by latrunculin B treatment, indicating that the defect in signaling lies prior to Akt/PKB activation. In summary, our data support the hypothesis that the actin network plays a role in organization of the insulin-signaling complex but is not required for vesicle trafficking and/or fusion.     

DIMETHYL SULFOXIDE-INDUCED REVERSION OF SEVERAL FEATURES OF POLYOMA TRANSFORMED BABY HAMSTER KIDNEY CELLS (BHK-21) : Alterations in Growth and Morphology

Cells of a polyoma virus transformed clonal line (Cl-I) of baby hamster kidney fibroblasts (BHK-21) were grown in medium containing 2 percent dimethylsulfoxide (DMSO). Unlike the untransformed BHK-21 cells, Cl-I cells adapted to replication in the presence of DMSO, and they exhibited a rapidly reversible phenotypic reversion of a number of properties characteristic of the transformed state. Restoration of density dependent growth inhibition with accumulation of cells in the G₁ phase of the cell cycle occurred and was associated with restoration of contact dependent behavior and with reversion of histological and ultrastructural features towards those which characterize untransformed cells. Concomitantly, Cl-I cells grown in 2 percent DMSO lost the ability to form colonies in semisolid medium. The data presented suggest that DMSO alters the expression of cellular functions which were altered as a result of viral transformation and which may be involved in cell tumorigenicity.     

The effect of CaCl2 and verapamil on the binding of cisplatin to cultures of normal and transformed human fibroblasts

Normal and transformed human fibroblasts were treated for either 1 sec or 1 h with the antitumor drug cis-dichlorodiamine platinum (cisplatin). The dose response of drug binding and cell survival was determined for cells treated with the drug in the presence or absence of 3.0 mM CaCl2. The levels of drug initially bound to both cell types was similar and was not affected by the presence of Ca2+. The dividing non-transformed cells were most sensitive to killing by short treatment with cisplatin compared to the transformed cells or the confluent non-transformed cultures. After 1 h of cisplatin treatment, the levels of drug bound to the cells were significantly less than that recovered after the shorter treatment. This time-dependent loss of cisplatin was inhibited both by CaCl2 and by the calcium channel blocking agent, verapamil. The higher levels of cisplatin bound after 1 h in the presence of these agents, however, did not in all cases result in decreased survival; the effects were dependent on cell type and on whether the cells were dividing or confluent. Analysis of cisplatin binding to cell cultures indicated that initially the cisplatin was weakly attached to the pericellular and substratum attached material but that with time, the drug bound to this material decreased. This time-dependent removal from the extracellular matrix was much less in the transformed cell cultures and was inhibited by calcium. We propose that the major site of interaction of cisplatin with these cells is in the extracellular matrix and with time the cultures alter their extracellular matrix to decrease this binding. This removal process appears to involve calcium or calcium transport since CaCl2 and verapamil both block these changes.     

Cognate interactions between helper T cells and B cells. II. Dissection of cognate help by using a class II-restricted, antigen-specific, IL-2-dependent helper T cell clone

In order to determine the involvement of T-B cell contact vs lymphokine production in mediating B cell cycle entry and progression, Th cell clones "defective" in lymphokine production were cloned. Th-3.1 is one such clone that required IL-2 to produce significant levels of IL-4 and IFN-gamma. Unlike conventional Th clones, Th-3.1 induced B cell proliferation only in the presence of Ag and IL-2. In contrast to the absolute requirement of IL-2 for Th-3.1-induced B cell proliferation, IL-2 was not required for the formation of stable Th-3.1-B cell conjugates or Th-3.1-induced B cell entry into the G1 phase of the cell cycle. In the absence of IL-2 and under conditions that promoted Th-B cell interactions, Th-3.1 induced 10 to 20% of resting B cells to enter G1. B cell entry into the cell cycle was not inhibited by anti-lymphokine mAb or promoted by exogenous lymphokines, suggesting that endogenous lymphokine activity was not required for Th-3.1-induced G0 to G1 transition. The data suggested that the IL-2-independent induction of B cells into G1 by Th-3.1 was a cell contact-dependent event. Direct proof that Th-3.1-B cell contact was necessary for B cell cycle entry was provided by comparative in situ analysis of the RNA synthetic activity and the RNA content of B cells that were in physical contact with Th-3.1 or not in contact with Th-3.1. In situ autoradiography of RNA synthesis illustrated that a high frequency of B cells in contact with Th-3.1 expressed heightened RNA synthetic activity, whereas "bystander" B cells were less frequently induced into cycle. In situ laser cytometry of B cell size and total RNA content showed that B cells in physical contact with Th-3.1 had a higher RNA content and were larger than "bystander" B cells present in the same microcultures. This model system has allowed the dissection of T cell help into IL-2-dependent and IL-2-independent phases. Early cell contact-dependent events and B cell cycle progression into G1 were IL-2 independent, whereas the production of lymphokines (IL-4, IFN-gamma) by Th-3.1 and Th-3.1-induced B cell proliferation was IL-2 dependent.     

Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes.

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.     

Biochemical characteristics of normal and virally transformed mouse cell lines

Normal and virally transformed mouse cells respond differently in vitro to high concentrations of potassium. A higher potassium concentration is required to inhibit multiplication of SV40 transformed 3T3 cells to the same extent as that of normal 3T3 cells. This potassium effect correlates to specific activity of Na-K dependent ATPase in membranous fraction, normal 3T3 cells having the higher enzyme activity being more sensitive to potassium than SV40-3T3 cells which have the lower specific activity of the enzyme. Contact inhibition of growth and changes of concanavalin A binding sites which are characteristics of viral transformation influence specific activities of Na-K dependent ATPase and of adenyl cyclase. Incubation with trypsinized concanavalin A causes SV40-3T3 to show contact inhibition of growth and at the same time, higher specific activities of both enzymes than the observed in untreated cells. Cellular content of cyclic 3′,5′-AMP of contact inhibited 3T3 is higher than that in transformed SV40-3T3, but no difference is detectable in ATP content.     

Inhibition of biological effects of bromodeoxyuridine by deoxycytidine: correlation with decreased incorporation of bromodeoxyuridine into DNA.

The effects of 5-bromodeoxyuridine (BrdU) on pigmentation, contact inhibition, cell morphology, and tumorogenicity of Syrian hamster melanoma cells are inhibited in the presence of deoxycytidine (dC). The inhibition of these biological effects of BrdU by dC is correlated with a decrease in the incorporation of BrdU into nuclear DNA. The results suggest that the intracellular changes resulting from the addition of dC to cells in the presence of BrdU are comparable to those resulting from a decrease in the concentration of BrdU in the medium.     

Role of β-GP-derived pi in mineralization via ecto-alkaline phosphatase in cultured fetal calvaria cells

The permissive effect of β-GP on mineralization in cultured rat fetal calvaria cells was investigated in relationship with phosphohydrolase activity of ecto-ALP at physiological pH range. β-GP present in the culture medium for 8 days exerted a stimulatory effect on ⁴⁵Ca incorporation into matrix cell layers while the ecto-ALP activity level measured on intact cells with a saturating concentration of p cells grown either in the presence or absence of β-GP. In both types of cultures, β-GP addition inhibited pNPP hydrolysis in a competitive and reversible manner and increased Pi concentration in the medium. The dose dependency of the effect of β-GP on ⁴⁵Ca incorporation and generation of Pi was similar (kϕ = 3 mM). Levamisole, but not dexamisole, inhibited both pNPP and β-GP hydrolyses, which were likely catalyzed by the same ecto-enzyme. The rate of ⁴⁵Ca incorporation into matrix cell layers, which was high (0.90 μmol/4h/mg cell protein) in cells grown in the absence of β-GP, was inhibited by 50% by levamisole. In cells grown in the absence of β-GP, the ⁴⁵Ca incorporation rate increased progressively after β-GP addition, reaching after 12 h the value of cultures grown in the presence of β-GP, the increase being totally inhibited by levamisole. In both types of cells, addition of exogenous Pi at concentrations corresponding to medium levels of β-GP-derived Pi rapidly led to high ⁴⁵Ca incorporation rate which was unaffected by levamisole. β-GP removal from cultures grown in its presence reduced by 50% the ⁴⁵Ca incorporation rate which recovered the initial value after exogenous Pi addition independently of levamisole presence. Thus, mineral deposition did not affect the level and catalytic efficiency of ecto-ALP to hydrolyze β-GP in cultured fetal calvaria cells, yet it influenced the β-GP-stimulatory effect on mineralization so as to render this process not sensitive to high medium Pi levels. © 1996 Wiley-Liss, Inc.     

Glucose repression of enterotoxins A, B and C and other extracellular proteins in staphlyococci in batch and continuous culture.

The production of enterotoxins, lipase and total extracellular protein by four strains of Staphylococcus aureus grown in batch culture at a controlled pH of 6.5 in a completely defined medium was markedly reduced by glucose or glycerol constantly maintained at 0.I M. A concomitant increase in the production of deoxyribonuclease, up to 13-fold, showed however that not all extracellular proteins are under the same control mechanism. The presence of glucose and glycerol in the medium also resulted in a rapid increase in the specific growth rate. However, growth of S. aureus s6 in Mgilimited continuous culture showed that glucose repression of enterotoxin B when the growth rate was held constant was more than twice that in batch culture. Therefore glucose repression can occur independently of an increase in growth rate. The specific rate of production of enterotoxin B, lipase, deoxyribonuclease, beta-haemolysin and total extracellular protein by S. aureus s6 increased as the growth rate increased from 0.07 to 0.24 h-1. Non-replicating cells grown in the absence of glucose produced considerable amounts of enterotoxin, and production was not repressed by the presence of glucose in the resuspension medium. In contrast, no enterotoxin B or C was obtained from nonreplicating cells grown in the presence of glucose. Chloramphenicol completely inhibited enterotoxin production by non-replicating cells, indicating that synthesis of new protein was required.     

Effect of yeast growth conditions on yeast-mycelial transition in Candida albicans

When grown and induced to form germ tubes in liquid defined media, yeast cells of Candida albicans must reach stationary phase before acquiring ability to carry out the yeast-mycelial transition. This study examined the effect of the carbon source utilized for yeast growth on the inducibility of stationary phase yeast. When grown to the same stationary phase cell density as glucose cultures, cultures grown on citrate were fully inducible while cultures grown on galactose and mannose showed a small reduction. Cultures grown on ethanol were reduced 80% in morphological conversion. When glucose grown cells were induced in the presence of these carbon sources, hexoses supported full induction while ethanol reduced induction 80%. Induction in the presence of carboxylic acids was similar to induction in the absence of added carbon source. When induced on the same source used in yeast growth, germ tube formation was reduced for all carbon sources except hexoses. When induced in the absence of added carbon source, yeasts grown on citrate and ethanol were inhibited 80-100%. Cultures starved for glucose were more inhibited than cultures starved for NH4Cl when induced without added carbon source. These observations suggest that the metabolic state of the stationary phase cell is an important factor in the ability to respond to conditions inducing germ tube formation.     

Effects of amino acids on the amidation of polyaromatic carboxylic acids by Bacillus cereus

The soil bacterium Bacillus cereus Tim-r01 efficiently transformed polyaromatic carboxylic acids (PACA) such as 4-biphenylcarboxylic acid (4-BPCA), 4-biphenylacetic acid, and 4-phenoxybenzoic acid into their corresponding amides. The amidation activity was expressed at 37 degrees C (pH 7-8) in the presence of grown cells in nutrients under an aerobic atmosphere. Other strains of B. cereus, IFO 3001 and IAM 1229, also gave the amide from 4-BPCA. In phosphate-buffered saline (PBS), the addition of normal amino acids was essential, while sulfur-containing amino acids such as methionine and cysteine drastically inhibited the amidation. Tracer experiments using N-15-isoleucine and N-15-alanine showed that the nitrogen atom of the amide came from an amino group of amino acids but not from ammonia or alkylamines.     

Tolerance to bromodeoxyuridine in a thymidine-requiring strain of Bacillus subtilis

Mutations which allow tolerance to 5-bromo-2'-deoxyuridine (BUdR) in a thymidine (TdR)-requiring strain of Bacillus subtilis have been examined. Differences in sensitivity to BUdR existed between isogenic strains harbouring the mutations. Those mutations originally isolated as BUdR-tolerant also bestowed tolerance to 5-bromouracil and vice versa. The strain exhibiting the greatest tolerance to BUdR maintained a normal rate of replication in the presence of BUdR whereas the parent strain did not, but the tolerant strain incorporated less analogue into DNA than the parent strain. The basis of the tolerance mutation appeared to lie at the point of uptake of the analogue into the cell as the tolerant mutant preferentially took up TdR over BUdR into whole cells. DNA polymerase activity measured in vitro did not distinguish between TdR and BUdR in either the parent or the mutant strain and although TdR kinase activity showed a preference for TdR over BUdR as a substrate, the extent of discrimination was similar in both strains.     

Cytoplasmic membrane polarization in Gram-positive and Gram-negative bacteria grown in the absence and presence of tetracycline

The ability of numerous diverse compounds and ions to cross the bacterial cytoplasmic membrane by diffusion and active transport is highly dependent on cytoplasmic membrane fluidity, which can be measured using fluorescent probes to estimate membrane polarization values. However, membrane polarization data are lacking for most bacterial species. The cytoplasmic membrane polarization values for Arthrobacter sp. ATCC 21908, Bacillus cereus NRC 3045, Pseudomonas fluorescens R2F, Pseudomonas putida NRC 2986 and Escherichia coli C600 bacterial cells were spectrofluorometrically measured over a temperature range from 10 to 50 degrees C, and in the absence and presence of 1 microg/ml tetracycline, using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to obtain new information on their membrane fluidity. At an assay temperature of 10 degrees C, E. coli cells grown in the absence of tetracycline exhibited the highest cytoplasmic membrane polarization value (least fluid membrane) of 0.446, followed by values of 0.392, 0.371, 0.344 and 0.293, respectively, for B. cereus, Arthrobacter sp., P. fluorescens and P. putida. At an assay temperature of 30 degrees C, the polarization values ranged from 0.357 to 0.288 for cells grown in the absence of tetracycline, regardless of the species. B. cereus grown in the presence of 1 microg/ml tetracycline had lower polarization values than when grown in the absence of this antibiotic at all assay temperatures. Regardless of the absence or presence of 1 microg/ml tetracycline in the growth medium, all bacterial species generally exhibited a more fluid membrane as the assay temperature increased from 10 to 50 degrees C. To our knowledge, these are some of the first cytoplasmic membrane polarization values reported for these Gram-negative and Gram-positive bacteria over a broad temperature range and also for cells grown in the presence of tetracycline.     

Microfilament-disrupting Clostridium difficile toxin B causes multinucleation of transformed cells but does not block capping of membrane Ig

The effects of Clostridium difficile toxin B on some actin-dependent cellular functions were studied. A three-day incubation of intoxicated B-lymphocytes and transformed 3T3 fibroblasts resulted in dose-dependent multinucleation. Using vimentin-negative Daudi cells we showed that this effect of toxin B does not involve vimentin. As DNA and protein syntheses are not impaired in the cells used, the results suggest that toxin B has an effect on the actin-containing contractile ring during mitosis, in a manner similar to that of the microfilament-disrupting agent cytochalasin B. Toxin B is the first bacterial toxin shown to have this effect. It was also found that the capping of surface IgM on B-lymphocytes was not inhibited by toxin B, whereas cytochalasin B did inhibit capping. These results suggest that capping is dependent on a specific membrane-associated actin structure, which is not affected by toxin B.     

Oncogene-induced transformation of a rat embryo fibroblast cell line is enhanced by tumor promoters.

Rat embryo fibroblast cell line 6 was transfected with plasmid pT24, which contains the activated human bladder c-Ha-ras oncogene, and the cells were grown continuously in the absence or presence of the tumor promoters 12-O-tetradecanoyl phorbol-13-acetate (TPA) or teleocidin. The presence of TPA or teleocidin led to a 6- to 14-fold increase in the number of morphologically transformed foci. No transformed foci were seen when rat 6 cells were transfected with the normal c-Ha-ras oncogene in the absence or presence of TPA, or in cells simply treated with TPA or teleocidin. Enhancement of pT24-induced foci was seen even when the addition of TPA was delayed until day 16. In transfection studies with the drug resistance genes gpt and neo, TPA and teleocidin did not increase the number of Gpt+ or Neo+ colonies. When rat 6 cells were cotransfected with pT24 and neo genes and grown in the absence or presence of TPA, the presence of TPA did not increase the yield of Neo+ colonies but caused a fivefold increase in the number of Neo+ colonies that displayed a transformed morphology. Southern blot analyses of DNAs obtained from these clones indicated that TPA treatment did not influence the extent of integration of either the pT24 or neo gene. DNA samples from all of the morphologically transformed cells displayed a characteristic 2-kilobase SacI fragment homologous to pT24 DNA and expressed relatively high levels of the corresponding mRNA. Our findings indicate that in this system tumor promoters do not simply enhanced the process of DNA transfection per se. Thus, this model system may be useful for analyzing synergistic interactions between tumor promoters and activated oncogenes during multistage carcinogenesis. It may also serve as a simple screening test for detecting new tumor promoters.     

Conditioned medium from endothelial cell cultures can restore the normal phenotypic expression of vascular endothelium maintained in vitro in the absence of fibroblast growth factor

Bovine vascular endothelial cells continuously maintained and grown in the presence of FGF adopt at confluence the configuration of a cell monolayer composed of contact-inhibited cells which do not overgrow each other and which are highly flattened and closely apposed. Such cultures exhibit structural and morphological characteristics similar to those observed with their in vivo counterparts. These include the production of an extracellular matrix consisting mostly of basement membrane collagen and fibronectin localized exclusively beneath the cell monolayer, but not on top of it, as well as a nonthrombogenic, blood-compatible apical cell surface. Removal of fibroblast growth factor (FGF) from adult bovine aortic endothelial cell (ABAE) cultures results within three passages in the loss by the cells of their characteristic contact-inhibited morphology. The cells, which during their logarithmic growth phase divide with a greatly increased doubling time, become larger and more elongated. Confluent cultures, instead of adopting the morphology of a contact inhibited cell monolayer, are now composed of overgrowing cells. Parallel with the morphological alterations taking place within the culture, the cells also lose the polarity of cell surfaces characteristics of the vascular endothelium. Formation of an extracellular matrix composed primarily of fibronectin and collagen types I, III, and IV is observed on both the apical and basal cell surfaces. Platelets which previously did not bind to the apical cell surface now become capable of binding to it. CSP-60, a major cell surface protein present in highly confluent and contact-inhibited vascular endothelial cell cultures, can no longer be detected. Exposure of confluent endothelial cell cultures, maintained in the absence of FGF to medium conditioned by cells which had been grown in the presence of FGF, but maintained in its absence upon reaching confluence led, within four to eight days, to a reversion of the altered phenotype. This medium has little or no mitogenic activity and retains a full activity in the absence of serum or after depletion of its fibronectin content by affinity chromatography on a gelatin-Sepharose column. Cultures which were previously composed of cells growing in multiple layers reorganized into a single cell monolayer composed of closely apposed and highly flattened cells. The cultures thereby regained the contact-inhibited morphology characteristic of the vascular endothelium. Concomitant with this cellular reorganization, the extracellular matrix disappeared from the apical cell surface, the cells regained their nonthrombogenic properties, and CSP-60 reappeared as one of the major cell surface proteins. These results suggest that vascular endothelial cells secrete a soluble factor(s) which can restore the normal morphology and function lost following removal of FGF from the medium. Such a factor(s) may be involved in maintaining the differentiated state of the vascular endothelium.     

T cell-dependent activation of B cell proliferation and differentiation by immobilized monoclonal antibodies to CD3

The capacity of mAb directed at the CD3 molecular complex (64.1) to induce T cell-dependent B cell proliferation and differentiation was examined. Coculture of B cells with mitomycin C-treated T4 cells (T4 mito) stimulated by immobilized 64.1 resulted in marked B cell proliferation and Ig-secreting cells (ISC) generation in the absence of any additional stimulation. The magnitude of the B cell responses induced by immobilized 64.1-stimulated T4 mito was far greater than that induced by other stimuli, such as Staphylococcus aureus plus factors produced by mitogen-activated T cells, PWM, or soluble 64.1. The induction of maximal B cell responsiveness required direct contact between activated T cells and responding B cells. Of note, immobilized 64.1 also induced B cell proliferation and ISC generation in the presence of mitomycin C-treated T8 cells. By contrast, immobilized 64.1 stimulated T4 or T8 cells that had not been treated with mitomycin C induced very modest ISC generation and suppressed B cell responses supported by T4 mito even in the presence of exogenous IL-2 or factors produced by mitogen-activated T cells. The interactions between T and B cells in these cultures not only induced B cell responses, but also enhanced the production of IL-2 by activated T cells. Increased IL-2 production was facilitated when culture conditions afforded the opportunity for contact between B cells and activated T cells. These results indicate that the establishment of interactions between B cells and anti-CD3-stimulated T4 or T8 cells provides all of the signals necessary for proliferation and differentiation of B cells without other stimuli and also augments the production of lymphokines by the activated T cells. The data emphasize the role of Ag-nonspecific interactions between B cells and T cells in promoting polyclonal responses of both cell types.