Cell growth cycle block of T cell hybridomas upon activation with antigen.

Stimulation of antigen-specific T cell hybridomas with the appropriate antigen/MHC combination, at concentrations that resulted in the secretion of the lymphokine interleukin 2, resulted in a dose-dependent decrease in both [3H]thymidine incorporation and cell growth. Flow cytometric studies demonstrated that stimulation with antigen resulted in a cell cycle block that was most evident at the G1/S border, and mixing studies revealed that bystander T cells of different antigen specificities were not affected. For at least the large majority of T cells, the G1/S cell cycle block appeared to be irreversible after 24 h of exposure to antigen. This cell cycle block may be useful as a rapid and quantitative measure of T cell hybridoma activation, as a means of selecting T cell hybridomas that have functional alterations in the reception of stimulatory signals, and may serve as a model of the induction of tolerance in immature T cells.

Chick embryonic skin as a rapid organ culture assay for cellular neoplasia.

We used chick embryonic skin (CES) in organ culture to assess the neoplastic potential of a variety of cultured human and nonhuman cell lines. Cells derived from cancer tissues grew in CES and formed tumors in nude mice while cells derived from normal tissues grew in neither system. The CES proved to be more sensitive than the nude mouse when used to assay SV40 transformed human cells; each of four such lines grew in CES while only one of the four lines grew and formed tumors in nude mice. In addition, the patterns of invasion by inoculated cells can be easily studied in the CES. These results suggest that CES in organ culture offers an inexpensive, rapid, and reliable alternative to the nude mouse as a tumorigenicity test.

Antibody-dependent cell-mediated cytotoxicity: detection by automated flow cytometry with ultramicro techniques.

Antibody-dependent cell-mediated cytotoxicity can be measured with as few as 1000 leukocytes with an automated flow cytometry technique.

Effects of 8-chloroadenosine 3',5'-monophosphate and N6-benzyl-cyclic adenosine 5'-monophosphate on cell cycle kinetics of HL-60 leukemia cells.

Site-selective cyclic AMP (cAMP) analogues have been shown to inhibit growth and induce differentiation in several human leukemia cell lines. However, detailed studies of the effects exerted by cAMP analogues on cell cycle kinetics have been lacking. We have examined the effects of 8-Cl-cAMP and N6-benzyl-cAMP on the cell cycle kinetics of the HL-60 human promyelocytic leukemia cell line. A cell cycle study was performed by univariate DNA analysis after 24-72 h of treatment with noncytotoxic concentrations of 8-Cl-cAMP and N6-benzyl-cAMP capable of inducing 50-60% growth inhibition in these cells. HL-60 cells treated with 5 microM 8-Cl-cAMP showed no significant change in the cell distribution in the cycle as compared to the untreated control cells, whereas the treatment with 10 microM N6-benzyl-cAMP transiently increased the percentage of cells in the G0/G1 phase after 48 h, followed by a partial recovery at 72 h. Combined treatment with low doses of 8-Cl-cAMP and N6-benzyl-cAMP, each of which alone produced 20% growth inhibition, exerted a growth inhibitory effect of 65% and delayed increase of the G0/G1 phase by 72 h. To better understand the cell cycle effects induced by 8-Cl-cAMP, flow cytometric analysis of bromodeoxyuridine incorporation was also performed. 8-Cl-cAMP treatment exhibited a slowing down of the cell cycle; thus, the delayed appearance of the G0/G1 cell accumulation after combined treatment could be due to this effect of 8-Cl-cAMP on the HL-60 cell cycle. At a toxic dose, 8-Cl-cAMP brought about a G2M block, whereas N6-benzyl-cAMP brought about an increase of the G0/G1 compartment. G2M block produced by toxic doses of 8-Cl-cAMP was not related to its adenosine metabolite since 8-Cl-adenosine did not produce any specific block in the cell cycle. Our results show, for the first time, that these site-selective cAMP analogues could affect cell cycle kinetics at different points. These data may provide the basis for combination treatments involving cAMP analogues and other agents in the treatment of human leukemia.

The Drosophila gene escargot encodes a zinc finger motif found in snail-related genes.

Two independent P-element enhancer detection lines were obtained that express lacZ in a pattern of longitudinal stripes early in germband elongation. In this paper, molecular and genetic characterization of a gene located near these transposons is presented. Sequence analysis of a cDNA clone from the region reveals that this gene has a high degree of similarity with the Drosophila snail gene (Boulay et al., 1987). The sequence similarity extends over 400 nucleotides, and includes a region encoding five tandem zinc finger motifs (72% nucleotide identity; 76% amino acid identity). This region is also conserved in the snail homologue from Xenopus laevis (76% nucleotide identity; 83% amino acid identity) (Sargent and Bennett, 1990). We have named the Drosophila snail-related gene escargot (esg), and the region of sequence conservation common to all three genes the 'snailbox'. A number of Drosophila genomic DNA fragments cross-hybridize to a probe from the snailbox region suggesting that snail and escargot are members of a multigene family. The expression pattern of escargot is dynamic and complex. Early in germband elongation, escargot RNA is expressed in a pattern of longitudinal stripes identical to the one observed in the two enhancer detection lines. Later in development, escargot is expressed in cells that will form the larval imaginal tissues, escargot is allelic with l(2)35Ce, an essential gene located near snail in the genome.

Detection of antibody-dependent cell-mediated cytotoxicity by automated flow cytometry. Comparison to a chromium release assay and characterization of the effector cell subpopulation.

Antibody-dependent cell-mediated cytotoxicity (ADCC) against chick red blood cells (CRBC) can be detected by flow cytometric (FCM) analysis of cellular DNA content. When compared to a standard chromium release assay FCM analysis shows several advantages: (1) equivalent cytotoxicity can be detected after 1 h compared to 4 h for 51Cr; (2) equivalent cytotoxicity can be seen at a 5-fold lower effector-to-target ratio; and (3) no radiolabeling is needed. When mouse spleen cells were fractionated based on adherence to the plastic, adherent cells showed the highest ADCC by both FCM and 51Cr release.

Cell transplantation and Federal regulation.

Federal regulation of tissues and organs has proceeded slowly, with main emphasis on safety of the procured material. More recently with the development of somatic cell therapies, the Food and Drug Administration has issued some guidance documents that establish that some classes of cells that are manufactured will be subject to not only safety but efficacy requirements. Cell transplantation presents several aspects that are similar to somatic cell therapies, and the purpose of this presentation is to explore those relationships.

Prognostic significance of DNA ploidy in carcinoma of prostate.

Flow cytometry was used to measure the DNA content in archived paraffin-embedded human prostatic cancer tissue for 69 patients with known outcomes that presented between 1975 and 1982. Of these, 51 patients had clinically localized lesions and were surgically staged prior to radical prostatectomy, while 18 patients presented with advanced Stage D2 disease. Thirty-six of 37 (97.3%) pathologic Stage B lesions were diploid. In contrast, the majority (72.2%) of patients with metastatic disease had aneuploid tumors. The average Gleason grade for aneuploid tumors was 8.2 +/- 1.98 versus 5.5 +/- 1.89 for diploid tumors (p less than 0.01). For 51 patients with clinically localized tumors, 13.9 percent of diploid tumors with a low Gleason sum (2 to 6) had extracapsular spread of tumor or regional lymph node involvement compared with 83.3 percent of aneuploid tumors with high Gleason scores (7 to 10). The addition of DNA ploidy to degree of glandular differentiation may enhance the prognostic evaluation of prostatic tumors and eventually improve our ability to select patients who are likely to benefit from radical prostatectomy.

Tumorigenicity testing: past concerns, future problems.

Tumorigenicity testing has been an integral part of cell substrate characterization. With the combination of recombinant technology and traditional cell culture, we have entered an era in which many of the principles developed with vaccine production may need to be modified and new ones developed. Models of tumorigenicity are reviewed in the light of current knowledge; the applicability and necessity of such testing in new areas such as somatic cell therapy will also be presented.

Receptors for interleukin (IL)-4 do not associate with the common gamma chain, and IL-4 induces the phosphorylation of JAK2 tyrosine kinase in human colon carcinoma cells.

We have previously reported on the expression of interleukin-4 receptors (IL-4R) on many human epithelial cancer cells; however, the binding characteristics, structure, function, and signal transduction through the IL-4R in cancer cells is not known. IL-4 binding characteristics were determined in human colon carcinoma cell lines by a 125I-IL-4 binding assay, which demonstrated that the HT-29 and WiDr colon cancer cell lines expressed high affinity IL-4R (Kd = 200 pM). Cross-linking experiments revealed a major band of 140 kDa and a broad band at 70 kDa. While the common gamma chain of IL-2R is associated with IL-4R in immune cells and is similar in size to the 70-kDa protein, this chain was not expressed in these colon cancer cells. Interestingly, IL-13, which has many functions similar to IL-4, inhibited 125I-IL-4 binding to both the 140- and 70-kDa molecules. Next, we investigated the mechanism of IL-4-induced signal transduction in colon cancer cells. After stimulation with IL-4, a 170-kDa band was primarily phosphorylated within 1 min of exposure and was identified as insulin receptor substrate-1. In addition, by immunoprecipitation assay, three other phosphorylated bands were identified as JAK1, JAK2, and Tyk2 tyrosine kinases. The phosphorylation of JAK1 and JAK2 was induced by IL-4 stimulation; however, Tyk2 was constitutively phosphorylated, and IL-4 treatment further augmented this phosphorylation. The kinetics and in vitro kinase assays demonstrated that JAK1, JAK2, and Tyk2 were phosphorylated within minutes and that JAK1 and JAK2 were activated after IL-4 exposure. Contrary to observations in immune cells. JAK3 mRNA was neither detected in colon cancer cells nor did IL-4 treatment cause phosphorylation of JAK3. These data indicate that in colon carcinoma cells JAK1, JAK2, Tyk2, and insulin receptor substrate-1 are phosphorylated after IL-4 stimulation. In addition, as is the case in lymphoid cells, IL-4 activated and phosphorylated signal transducers and activators of transcription (IL-4-STAT or STAT-6) protein in both colon cancer cell lines. These results indicate that the IL-4R complex is composed of different subunits in different tissues and shares a component with the IL-13R complex. In addition, we demonstrate for the first time that like its family members (e.g. IL-3 and GM-CSF), IL-4 can phosphorylate and activate JAK-2 kinase.

Colony-stimulating factor (CSF) controls proliferation of CSF-dependent cells by acting during the G1 phase of the cell cycle.

Proliferation of granulocyte/macrophage (GM) progenitor cells in soft agar cultures is dependent on the continuous presence of colony-stimulating factors (CSF). To elucidate this dependency we studied the effect of deprivation and readdition of CSF on the cell cycle kinetics of a GM-CSF-dependent murine mast/basophil cell line (PT-18). Flow cytometry and [3H]thymidine incorporation have been used to analyze the complete cell cycle. Removal of CSF from the culture medium resulted in accumulation of the cells in the G1 phase. Eighteen hours after removal of CSF, 85% of the cells were arrested in G1 phase. Readdition of GM-CSF to such quiescent cells was followed by progression of the cells from G1 into S phase with a lag period of 10 hr. A similar lag period was observed when cells released from G2+M arrest progressed, in the presence of CSF, to S phase via the G1 phase. These findings indicate that deprivation of GM-CSF does not move PT-18 cells out of the cycle to a G0 phase but rather arrests them at early G1 phase. Finally, we demonstrate that, for the cells to progress through the cell cycle, the requirement for the presence of GM-CSF is limited to the first 6 hr of the 10-hr duration of the G1 phase.

Measurement of DNA synthesis by flow cytometry.

Cells were stained for DNA content with Hoechst 33258 combined with propidium iodide and analyzed on an epi-fluorescence flow cytometer. Scatterplots of red versus blue fluorescence of control cells showed a straight line indicating that both dyes were measuring proportional amounts of cellular DNA. Scatterplots of cells incubated with the thymidine analog bromodeoxyuridine, however, showed a displacement from the control straight line that increased with longer exposure time to bromodeoxyuridine. These results suggest that bromodeoxyuridine incorporation in DNA can be detected by flow cytometry.

IL-13 induces phosphorylation and activation of JAK2 Janus kinase in human colon carcinoma cell lines: similarities between IL-4 and IL-13 signaling.

We have recently reported that IL-13R may share a component with IL-4R. Here we report that both IL-4 and IL-13 share signaling events in human colon carcinoma cell lines (HT-29 and WiDr). IL-13 caused rapid phosphorylation of the three out of four members of the known Janus family of kinases (JAKs). We show that JAK2 kinase is rapidly phosphorylated and activated in response to IL-13. Within 1 min of activation, JAK2 was phosphorylated, and peaked in 10 min. In addition, IL-13 phosphorylated insulin response substrate-1, IL-4R p140, JAK1, and Tyk2, but not JAK3 kinase. IL-4 also stimulated all three kinases and substrates, but unlike in immune cells, IL-4 did not involve JAK3 activation for its signaling in colon cancer cell lines. Furthermore, JAK2 associated with the IL-4R p140 before and after stimulation with IL-13. Both IL-13 and IL-4 induced phosphorylation of IL-4 STAT (STAT6) but not STAT1, STAT3, or STAT5. 125I-IL-13 did not bind to colon cancer cell lines, but unlabeled IL-13 competed for the binding of 125I-IL-4. Our data suggest that IL-13 utilizes IL-4R and its signaling pathway, and JAK2 may play an important role in the function of IL-4R and IL-13R in colon cancer cells.

Ultrastructural features of human galactosaemic fibroblasts: effects of different nutritional environments.

The ultrastructural characteristics of galactosaemic human fibroblasts exposed to media with various sugars were compared. Considerations of known biochemical pathways suggested that cells without the galactokinase enzyme and exposed to galactose would be functionally equivalent to cells in a medium without any sugar source. We found the ultrastructural characteristics of such cells to be consistent with that hypothesis. In addition, we unexpectedly found that homozygous galactosaemic fibroblasts, exposed to medium with glucose as the energy source, showed evidence of degeneration, such as autophagic vacuoles. Degenerative changes were not found in fibroblasts heterozygous for the galactokinase enzyme exposed to glucose. The implications and uses of these findings are discussed.

The cell cycle block and lysis of an activated T cell hybridoma are distinct processes with different Ca2+ requirements and sensitivity to cyclosporine A.

Stimulation of transformed T cells leads to both lymphokine secretion and inhibition of spontaneous growth. Studies performed with an Ag-specific T cell hybridoma demonstrated that growth inhibition is an early (within 1 h) manifestation of activation. Experiment in which extracellular Ca2+ was chelated or in which cyclosporine A was included indicated that activation-associated growth inhibition is a two-step process. The first phase is the establishment of a G1/S cell cycle block; it does not require extracellular Ca2+ and is not prevented by the addition of cyclosporine A. The second phase is cell lysis. It can be detected 4 to 6 h after activation, requires the presence of extracellular Ca2+, and is prevented when stimulation occurs in the presence of cyclosporine A. The observation that both Ca2+ depletion and cyclosporine A prevented IL-2 secretion at all time points indicates that the pathways leading to lymphokine secretion and the G1/S block diverge early in the course of the cellular response, and establish the cell cycle block as a distinct activation event with unique characteristics.

Heterogeneous expression of a murine B16 melanoma-associated antigen correlates with cell cycle.

A murine monoclonal antibody (MM2-3C6) that reacts with a B16 murine melanoma-associated membrane antigen was used to study the relationship of antigen expression to the cell cycle. Dual-parameter flow cytometric measurements of membrane antigen and DNA revealed that antigen-positive cells were present throughout the cell cycle. Peak antigenic expression was noted during the late log phase of the cell growth curve with negligible antigen-negative population. The emergence of a distinct antigen-negative population (30%-40%) was noted in the late stationary phase. Cell cycle analysis indicated that the negative subpopulation was restricted to the G0/G1 phase, thus, demonstrating antigenic heterogeneity within the tumor cell population. Cell sorting was performed to analyze the origin of such heterogeneity. Following two sequential sortings, the antigen-negative cells became antigenic upon reculture. Again, at the late stationary phase, a distinct antigen-negative population (30%-40%) emerged. The sorted antigen-positive cells showed flow cytometric profiles identical to the sorted antigen-negative population upon reculture. Therefore, in this murine model, it appears that antigen expression is cell cycle dependent and such expression seems to be associated with proliferation.

Inhibition of transformed T cell growth in vitro by monoclonal antibodies directed against distinct activating molecules.

Stimulatory of antigen-specific murine T cell hybridomas with the appropriate antigen has been shown to cause lymphokine secretion and inhibition of spontaneous cell growth. In this study, the effect of cellular activation on the growth of transformed T cells, of known or unknown antigen specificity, was explored with stimulatory monoclonal antibodies (mAb) that recognize nonclonally distributed T cell surface molecules. Anti-CD3 antibodies stimulated interleukin 2 (IL-2) secretion while they inhibited murine and human T cell tumor growth in vitro. Both responses required external cross-linking of the anti-CD3 antibodies. Activation via two molecules that are not physically associated with the T cell antigen receptor, Thy-1 and Ly-6, also inhibited transformed T cell growth while inducing IL-2 secretion. Notably, an anti-Thy-1 mAb that did not cause the transformed T cells to secrete lymphokines failed to affect their growth, and in fact blocked the growth inhibition induced by the stimulatory mAb. Regardless of which stimulating mAb was used, IL-2 production and cell growth were inversely proportional, manifesting similar antibody dose-response curves. Activation of a T cell hybridoma with stimulatory mAb resulted in rapid lysis, as evidenced by the release of 51Cr and lactate dehydrogenase. Cell cycle analysis demonstrated that cellular activation was accompanied by a cell cycle block between the G1 and S phases, and probably a slowing of the transit of cells already in S. These results demonstrate that the growth of a spectrum of neoplastic T cells, murine and human, can be inhibited by what are normally growth-promoting signals for non-transformed T cells.