Effects of ellipticine on cell survival and cell cycle progression in cultured mammalian cells

The effects of ellipticine [5,11-dimethyl-6H-pyrido(4,3-b)carbazole; NSC 71795] on cell viability, growth, and colony formation were investigated in suspension (Friend leukemia and L1210) and adherent [Chinese hamster ovary (CHO)]tumor cell systems as well as in mitogen-stimulated human peripheral blood lymphocyte cultures. Cell cycle progression and the terminal point of action of the drug were monitored by flow cytometry. Ellipticine was cytostatic for all cell lines tested, blocking cells in G2 phase following 24 hr constant exposure at concentrations in the range of 1.0 microgram/ml. A 10 times higher drug concentration was required to block cells in G2 if the cells were exposed for only 30 min to the drug followed by 23.5 hr culture in drug-free medium. Formation of CHO cell colonies was inhibited by 50% following exposure to ellipticine for 2 hr at 6.0 microgram/ml or for 24 hr at 0.3 microgram/ml. Fifty % cell kill in asynchronously growing Friend leukemia and L1210 cells was obtained following exposure to ellipticine for 24 hr at 2.0 microgram/ml and 1.15 microgram/ml, respectively, whereas human peripheral blood lymphocytes required 66 hr exposure to 1.0 microgram/ml to kill 50% of the cells. Phytohemagglutinin-stimulated lymphocytes were remarkably resistant to the cytotoxic effect of ellipticine but did display a dose-dependent inhibition of stimulation and accumulation in G2 whether the drug was added prior to our during active cell proliferation. Ellipticine, at cytostatic concentrations, had a marked effect on cellular RNA content. Friend leukemia cells, blocked in G2 by the drug, doubled their RNA content compared to control cells. L1210 and CHO cells, but not lymphocytes, also increased in RNA content following ellipticine treatment. Drug concentrations which blocked cells in G2 also led in the case of Friend leukemia and L1210 but not CHO cells to an increase in the proportion of cells with greater than 4C amounts of DNA.     

Production by undifferentiated myeloid leukemia cells of a novel growth-inhibitory factor(s) for partially differentiated myeloid leukemic cells

Mouse monocytic Mm-A cell line is a highly leukemogenic variant cell line of the monocytic and non-leukemogenic Mm-1 cell line, which developed spontaneously from mouse myeloid leukemia M1 cells. Growth-inhibitory factor (GI factor) for Mm-A cells was found in conditioned medium (CM) of differentiation inducer-resistant myeloblastic M1 cells (clone R-1). The R-1 cells were cultured with or without 2% calf serum for 2 days, and the CM was fractionated with 50% ammonium sulfate and used as the GI factor preparation (termed R1CM). When Mm-A cells were cultured with 5% (v/v) R1CM for 3 days, their growth was inhibited about 80%. This inhibition of Mm-A cell growth by R1CM was irreversible. This GI factor also inhibited the growth of M1 cells that had been pretreated with inducer and had expressed some differentiation-associated properties but still retained a proliferative capacity. In contrast, it scarcely inhibited the growth of untreated M1 cells. The GI factor inhibited the growth of other mouse monomyeloblastic leukemic WEHI-3B D+ cells pretreated with a differentiation inducer, retinoic acid, and mouse monocytic leukemia J774.1 cells. However, it did not affect the growth of human monocytic (U937 and THP-1) or myeloid (KG-1, ML-1, and HL-60) cell lines. These results suggest that GI factor produced by parent myeloblastic and inducer-resistant M1 cells preferentially inhibits the growth of mouse monocytic leukemia cells in intermediate stages of differentiation from myeloblastic leukemia cells to mature macrophages.     

Distribution and content of nuclear and cellular RNA among cell populations of acute lymphoblastic and nonlymphoblastic leukemia

The RNA content of intact cells and isolated nuclei of normal human lymphocytes and mononuclear cell populations (containing at least 50% blasts) from patients with acute lymphoblastic leukemia (ALL) and acute non-lymphoblastic leukemia (ANLL) was measured by flow cytometry based on metachromatic red luminescence of acridine orange-stained cells. Relative nuclear RNA (n-RNA) and cellular RNA (c-RNA) content was estimated in relation to luminescence of RNase-treated nuclei, which served as a standard. The mean values (+/- SE) of n-RNA were 22.6 +/- 3.2, 25.8 +/- 3.2, and 51.5 +/- 6.1 arbitrary units for normal lymphocytes, ALL, and ANLL cell populations, respectively. The mean values for c-RNA were 51.3 +/- 5.2, 71.9 +/- 11.3, and 128 +/- 13.4 for the same cell populations, respectively. The differences between normal lymphocytes or ALL and ANLL cell populations were statistically significant (t-test, with respect to both n- and c-RNA), while differences between normal and ALL populations were not. The proportions of n-RNA versus c-RNA were similar within all three types of cell populations. The intercellular variabilities with respect to n- and c-RNA among the G0/1 cell populations of all three types of cells were characterized by the coefficient of variation (CV) of the mean RNA and the third moment about the mean (MOM3). CV and MOM3 of c-RNA were significantly different between all three types of cell populations, whereas CV and MOM3 of n-RNA showed significant differences between control and leukemic cells. Thus, mean RNA, CV, and MOM3 of RNA on the cellular and nuclear levels of G0/1 cells discriminate normal lymphocytes, ALL lymphoblasts, and ANLL blast cells from each other fully on statistically significant levels.     

Hydroxyurea indices precommitment during retinoic induced HL-60 terminal myeloid differentiation: possible involvement of gene amplification

Control of terminal cell differentiation was studied in the HL-60 human promyeloctyic leukemia cell line. Retinoic acid is known to induce myeloid differentiation associated with GO arrest in these cells. In this case, onset of terminal differentiation occurs after an exposure period corresponding to two division cycles. This is preceded by acquisition of a precommitment memory state occurring by one division cycle. Cells in precommitment undergo accelerated onset of terminal differentiation upon reexposure to inducer. The present report shows that the precommitment state can be induced by a pulse exposure to hydroxyurea. While the hydroxyurea exposure does not itself induce terminal differentiation, the treated cells undergo accelerated onset of phenotypic differentiation and GO arrest upon exposure to retinoic acid. Thus a perturbation of S-phase specific cellular metabolism induces the early precommitment regulatory state in the course of induced HL-60 terminal myeloid differentiation. The results support a model in which initiation of a cellular program of terminal differentiation depends on an S-phase specific event associated with replication of cellular DNA and possibly involving gene amplification. Significantly, the results indicate that a conventional chemotherapeutic agent such as hydroxyurea can synergistically interact with a differentiation inducing agent such as retinoic acid. This indicates the significance of investigating the interaction between conventional S-phase specific chemotherapeutic agents and differentiation inducing agents as a potential treatment modality.     

Biochemical alterations during unperturbed suspension growth of L1210 cells

The following parameters were evaluated at several points throughout unperturbed suspension culture growth of L1210 cells: cell volume; DNA histograms; the mean content of cellular DNA, RNA, and protein; ribonucleoside and deoxyribonucleoside triphosphate pools; phosphoribosyl pyrophosphate; and the incorporation of glycine into purine bases. The cell volume, the incorporation of glycine into purine bases, and the intracellular pools of phosphoribosyl pyrophosphate and dexoyribunucleotides began to decrease significantly during the midportion of logarithmic cell growth. However, there was no significant change in the DNA content per cell during culture growth. The RNA, protein content, and ribonucleotides all demonstrated a biphasic pattern with the highest values obtained during the midportion of logarithmic growth followed by rapid decline as the culture approached plateau growth. These intracellular fluctuations in de novo synthesis and precursor pools were correlated with the variable intracellular accumulation of three fluoropyrimidines (5-fluorouracil, 5-fluorouridine, and 5-fluorodeoxyuridine) and their active metabolites (5-fluorouridine triphosphate and 5-fluorodeoxyuridylate). These studies were performed to demonstrate that multiple biochemical alterations occur during logarithmically growing suspension cell cultures and could result in misleading conclusions of experiments with antimetabolites unless these factors are considered in the context of the performed studies.     

Differentiated HL-60 promyelocytic leukaemia cells produce a factor inducing differentiation

The bipotential human promyelocytic leukaemia cell line HL-60 can be induced to differentiate into monocytic or granulocytic cells by treatment with 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) or dimethylsulphoxide (DMSO) respectively. Conditioned media (CM) from 1,25(OH)2D3- or DMSO-treated cells were able to induce monocytic differentiation in fresh HL-60 cells as measured by induction of non-specific esterase and macrophage surface markers. CM from 1,25(OH)2D3-treated cells also led to a dose dependent loss of proliferative capacity in soft agar colony assays. These effects were not due to a toxic effect of the CM or to residual inducer present in the CM. gamma-interferon and GM-CSF were apparently not responsible for these effects. CM from the human histiocytic lymphoma cell line U937 led to only a low level of induction of macrophage differentiation in fresh HL-60 cells. The defect in HL-60 leukaemic cells may therefore be at the level of induction of an autonomously-produced differentiation factor.     

Differentiation antigens of HL-60 promyelocytes during induced maturation

The human promyelocytic leukemic cell line HL-60 can be induced to mature monocytes and macrophages in vitro by lymphocyte-conditioned medium. We are reporting sequential changes in surface antigenic expressions, which are sensitive markers of the characteristic events in the process of cell differentiation. The promyelocyte membrane antigen, detected by a monoclonal antibody produced using HL-60 cells as an immunogen, was shown to be associated with immature myeloid cells and was used to determine HL-60 cell development. The expression of this membrane antigen, determined to have a molecular weight of 85,000 was lost early in the differentiation period. In the following stage, in which the promyelocytes developed into monocytic cells, a steady increase of cells bearing the OKM1 normal monocyte antigen was observed. When macrophages became predominant in the final culture period, the expression of the OKM1 antigen decreased. The usefulness of these differentiation antigens in studying cellular development is discussed.     

Effects of a prospective antitumor agent, 1,4-bis(2''-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate, on cultured mammalian cells

The effects of 1,4-bis(2'-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate (CBH; NSC 57198) on cell viability, growth, progression through the cell cycle, survival, and differentiation were investigated in suspension cultures of murine lymphocytic leukemia (L1210) and erythroleukemic (FL) cells and normal human lymphocytes stimulated with phytohemagglutinin (PHA) and in adherent cultures of Chinese hamster ovary (CHO) cells. CBH was equally cytotoxic toward stationary and exponentially growing CHO cells. Cell viability was diminished by 50% following 24 hr exposure to approximately 50 μg CBH per ml. Treatment of quiescent human lymphocytes for 24 hr with up to 100 μg CBH per ml did not appreciably diminish cell viability though the subsequent stimulation of such lymphocytes with PHA was inhibited in a dose dependent fashion. L1210, FL cells, and PHA stimulated human lymphocytes were equally sensitive to CBH, 50% inhibition of growth was obtained following 24 hr treatment with 25 μg CBH per ml. Incubation for up to 48 hr with CBH did not result in differentiation of FL cells to mature hemoglobin containing cells. Constant exposure of L1210 cells and PHA-stimulated human lymphocytes to 10-50 μg CBH per ml resulted in accumulation of cells in G₂ + M phase; higher drug concentrations resulted in cell arrest in mid to late S phase and G₂ phase. A short 1-hr pulse of the drug resulted in a transient accumulation of L1210 cells in S and G₂ phases. However, cells recovered from a short pulse of drug and by 48 hr, both cell proliferation and the cell cycle distribution appeared normal. A detailed analysis of cell cycle progression of L1210 cells in the presence of the drug indicated that the duration of G₂ phase was extended at low concentrations (10 μg/ml) while the transit of cells through S was retarded with subsequent accumulation in late S and G₂ phase at higher (50 μg/ml) concentrations. Concomitant with cell arrest in S and G₂ phase an increase in cellular RNA content indicating unbalanced growth was observed. This state of unbalanced growth was reversible in cultures exposed to a 1-hr pulse of up to 100 μg CBH per ml; cellular RNA content returned to control values by 48 hr. No effect on nuclear chromatin as assayed by acid denaturation was observed. Though the exact mechanism of drug action is not known, the data are not incompatible with the drug acting as an alkylating agent.     

Phorbol ester-induced differentiation of human T-lymphoblastic cell line HPB-ALL

12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, induced phenotypic differentiation in the human thymic acute lymphocytic leukemia cell line, HPB-ALL. Within 30 min of seeding in the presence of TPA, the cells formed a smooth round shape. After a 7-day exposure to TPA, most of the cells became smaller and reminiscent of large or atypical lymphocytes. Electron microscopic analysis evidenced morphological differentiation in TPA-treated HPB-ALL cells. Thymic antigens stained with monoclonal antibody OKT6 were dramatically reduced while Leu2a-positive cells were increased in the TPA-treated HPB-ALL cells. However, OKT3-positive cells did not appear in these TPA-treated cells for up to 7 days. Upon TPA-induced phenotypic differentiation, the growth rate of cells was significantly inhibited, their ability to incorporate DNA and RNA via 3H-labeled precursors was reduced, their ability to bind sheep red blood cell rosettes was significantly increased, and the proportion of terminal deoxynucleotidyl transferase-positive cells was decreased.     

Differentiation of HT-29 human colonic adenocarcinoma cells correlates with increased expression of mitochondrial RNA: effects of trehalose on cell growth and maturation.

The HT-29 human adenocarcinoma cell line has been used extensively in the study of colonic cell differentiation and colon cancer. We report here that substitution of glucose with trehalose (alpha-D-glucopyranosyl-alpha-D-glucopyranoside) depresses growth and promotes mucin-producing, goblet-like maturation of HT-29. An initial characterization of this process was made by analyzing several cDNA clones whose RNA templates were differentially expressed at elevated levels in cells grown in trehalose-containing medium. Seven of the 9 clones examined corresponded to 6 mitochondrial genes whose expression levels, relative to those from glucose-grown cells, ranged from approximately 3-fold for 16S rRNA to 8-23-fold for NADH dehydrogenase subunit 4. On the other hand, levels of mitochondrial DNA copy, measured by using NADH dehydrogenase subunit 4 cDNA as probe, were shown to be unaffected by trehalose treatment. Elevation of cellular NADH dehydrogenase subunit 4 RNA in HT-29 cultures grown in medium containing different components (sodium butyrate, galactose, no-sugar, glucose, cellobiose) generally correlated with depressed growth levels and specifically with increased numbers of mucin-producing cells present. Like butyrate, the sugar, trehalose, is an effective inducer of HT-29 differentiation, and may prove useful as a dietary therapeutic, and as a probe for elucidating mitochondrial involvement in colonic cell differentiation and transformation.     

Comparative effect of embryonic mouse fibroblasts (Balb/c-3T3) on the proliferation of hormone-dependent (T-47D) and hormone-independent (MDA-MB-231) human breast cancer cell lines

Summary The effects of cellular extracts (CE) and conditioned medium (CM) of embryonic mouse BALB/c-3T3 (clone A 31) cells on the proliferation and DNA content of hormone-dependent T-47D and hormone-independent MDA-MB-231 breast cancer cell lines were explored. After 6 days of culture, CE and CM provoke an intense proliferative effect in T-47D cells which correlates with DNA content. In contrast, in the MDA-MB-231 cells a significant inhibitory effect was observed. For both CE and CM the action was dose-dependent. In the T-47D cells, the CM can abolish the inhibitory effect provoked by the potent antiestrogen ICI 164,384. It is concluded that mouse embryonic BALB/c-3T3 cells contain factors which can stimulate or inhibit the growth of human mammary cancer cells.     

A new experimental model for in vivo studies on differentiation and proliferation of leukemic cells using a mouse myeloid leukemia aneuploid line

We developed an experimental model to investigate in vivo differentiation and proliferation of leukemia cells using mouse myeloid leukemia aneuploid cells (LL-2) and syngeneic SL mice. The LL-2 cells were near-tetraploid cells isolated from mouse myeloid leukemia cell line M1 (clone D501). In suspension culture, the LL-2 cells were myeloblastic and grew well like parent D501 cells, but were distinct from the parental cells due to the large size of their nucleus, double chromosome number and DNA content. The LL-2 cells as well as D501 cells could be induced to differentiate in vitro into mature macrophage-like cells by a protein inducer of differentiation. After transplantation of 4 X 10(6) LL-2 cells into the intraperitoneal cavity of syngeneic SL mice, most of them died of leukemia within 10 weeks. On microscopic examination of the peritoneal cells of the mice, the transplanted LL-2 cells were clearly distinguishable from normal host cells by the size of their nucleus. We determined the increase in the LL-2 cells in the peritoneal cavity by morphological examination of the large-sized LL-2 cells. Survival times of the mice inoculated with the LL-2 cells were prolonged by administrations of an inducer of differentiation, poly(I).poly(C). We found morphological changes in the peritoneal blastic LL-2 cells to mature macrophage-like cells after the serial administrations of poly(I).poly(C) to the recipient mice. Thus the aneuploid LL-2 cells that grow in syngeneic mice may be useful to study in vivo differentiation and proliferation of leukemia cells, and to develop a therapeutic strategy of leukemia using various treatments including differentiation inducers.     

Separation of spontaneously differentiating and cell cycle-specific populations of HL-60 cells

The human leukemia cell line HL-60 consists predominantly of abnormal promyelocytes. When grown in RPMI-1640 and 10% FCS between 5 and 10% of these cells spontaneously differentiate into more mature myeloid cells, becoming smaller in size and developing the ability to generate superoxide. Centrifugal elutriation was used to separate these G0 cells from the bulk of the cycling G1, S and G2M cells. These isolated differentiating cells are shown to be similar in size, DNA content, RNA content and NBT positivity not only to granulocyte induced HL-60 cells but also to human peripheral blood granulocytes. This methodology allows the study of differentiative vs proliferative processes through the quick one-step generation of homogeneous subpopulations of G0, G1, S and G2M cells.     

Modulation of the maturation of human leukemic promyelocytes (HL-60) to granulocytes or macrophages

The relationship between the effects of two types of inducers on the maturation of a line of human promyelocytic cells (HL-60) was studied. The dual potentiality of these promyelocytes was demonstrated by the ability of isolated colonies to differentiate into granulocytes, following induction by dimethylsulphoxide (DMSO) or express properties specific to macrophages following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). The differentiation process involved an irreversible step which occurred 48 h after exposure to DMSO, and a few h after exposure to TPA. This implies that the presence of the inducer in the culture medium is no more required for completion of the differentiation programme. Removal of the inducer prior to this step resulted in reversal of all the inducer-mediated effects. During the period of non-commitment the specific pathway of maturation was still undetermined; cells in which differentiation was initiated by exposure to DMSO were able to develop into macrophages after substitution of DMSO by TPA. Moreover, pre-exposure to DMSO and other inducers of granulocyte differentiation resulted in higher sensitivity to TPA, as indicated by the cell response to low TPA concentrations and more rapid expression of macrophage specific properties. These results suggest that the early stages in HL-60 differentiation are probably common to the granulocyte and the macrophage pathways.     

Evidence for cell cycle phase-specific initiation of a program of HL-60 cell myeloid differentiation mediated by inducer uptake

The question of whether the initial regulatory event, which directs an uncommitted precursor cell toward terminal differentiation, is cell cycle phase specific was examined using the human promyelocytic leukemia cell line, HL-60. While the HL-60 system does not reflect all of the features of normal hematopoiesis, it does provide a relatively well-defined in vitro experimental system which can be useful for examining aspects of the differentiation process. HL-60 cells were induced to undergo myeloid differentiation by retinoic acid. The subsequent differentiation kinetics of HL-60 populations initially enriched in different cell cycle phases was measured. This was compared to the cellular uptake of retinoic acid as a function of cell cycle position. If the initial differentiation-regulating event were cell cycle phase independent, then the kinetics of differentiation would be independent of the cell cycle status of the initial population. Flow cytometric cell sorting, based on cellular narrow angle and orthogonal light scatter intensity spectra, was used to select G1-enriched and S + G2 + M-enriched cell populations without pharmacological perturbation. These two populations were each induced to undergo myeloid differentiation with 10(-6) M beta-all-trans-retinoic acid. The kinetics of G1/0 arrest associated with terminal cell differentiation, as well as phenotypic differentiation, assayed by development of oxidative metabolism, was measured for both populations. The kinetics of differentiation differed for the two populations, indicating that the initial differentiation-regulating event was cell cycle phase specific. For both of the initial cell populations, significant phenotypic differentiation followed approximately 24 hr after enrichment in the relative number of S-phase cells. When exponentially proliferating HL-60 cells were exposed to a 1-hr pulse of 10(-5) M [3H]retinoic acid and then flow cytometrically sorted by DNA content, cells in late S + G2 + M had an approximately 10-fold higher uptake than cells in G1 or early S. The results indicate that cellular regulation of myeloid differentiation first becomes responsive to the inducer, retinoic acid, in S phase when uptake is enhanced.     

Crocin inhibits proliferation and nucleic acid synthesis and induces apoptosis in the human tongue squamous cell carcinoma cell line Tca8113

Background: Cancer chemoprevention is a proven effective strategy for oral squamous cell carcinomas (OSCCs). The present study was designed to investigate the effects of crocin, a potential chemopreventive agent, on growth and DNA and RNA content in a human tongue squamous cell carcinoma cell line, Tca8113. Methods: Tca8113 cells were treated with crocin for 24, 48, 72, and 96 h at concentrations of 0.1, 0.2, 0.4, and 0.8 mM. Tumor cell viability was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. In addition, Tca8113 cells were treated with 0.4 mM crocin and cytotoxic effects as an inducer of apoptosis were analyzed using flow cytometry. Furthermore, acridine orange (AO) staining and observation using laser scanning confocal microscopy (LSCM) were used to determine the effects of the drug on nucleic acid synthesis. Results: Crocin decreased Tca8113 cell viability and growth remarkably at 24, 48, 72, and 96 h, in a concentration-dependent manner (P<0.05). In addition, 0.4 mM crocin significantly induced both early and late apoptosis of Tca8113 cells. Moreover, the cellular DNA and RNA content was significantly downregulated by 0.4 mM crocin compared with the negative control (P<0.01). Conclusions: Our observations support the feasibility of applying crocin as a chemoprophylactic agent and treatment for OSCCs.     

Early activation of the proto-oncogene c-fgr during Epstein-Barr virus immortalization.

In an attempt to clarify the chronological relationships between Epstein-Barr virus (EBV) infection, B cell immortalization and c-fgr activation, we evaluated for the presence of EBV-determined nuclear antigen (EBNA), cellular DNA synthesis and expression of c-fgr-specific RNA following infection of human peripheral blood lymphocytes with B95-8 EBV. High expression of c-fgr was observed prior to EBNA detection and cellular DNA synthesis in EBV-infected cells. These results suggest that activation of c-fgr is an essential event during the early phase of EBV immortalization.     

Expression of retinoic acid receptor mRNA in hematopoietic cells

Retinoic acid (RA) has profound effects upon the proliferation and differentiation of many hematopoietic cells. The mechanism by which RA acts is unclear. Recently, several retinoic acid receptors (RAR) have been cloned. We studied expression of RAR-alpha mRNA by RNA blots in hematopoietic cells blocked at different stages of differentiation. All hematopoietic cells expressed RAR-alpha mRNA (3.4, 4.5 kb) including KG-1 (myeloblasts); HL-60 (promyelocytes); ML3, THP-1, U937 (myelomonoblasts and monoblasts); K562 (erythroblasts); and S-LB1 (T-lymphocytes). In addition, transformed cells from four non-hematopoietic tissues also expressed RAR-alpha mRNA. Steady-state levels of RAR-alpha mRNA were not affected by induction of terminal differentiation of HL-60 cells to either granulocytes or macrophages. Furthermore, both actively proliferating and resting lymphocytes from the same individuals expressed equal concentrations of RAR-alpha mRNA. Taken together, data suggest that level of expression of RAR-alpha mRNA is not related to cellular proliferation. We also showed that exposure to ligand (all-trans retinoic acid) did not change levels of RAR-alpha mRNA in three different cell types. Half-life of RAR-alpha mRNA was short (0.7 h) as determined by measuring decay of message after addition of actinomycin D. Consistent with this finding, accumulation of RAR-alpha mRNA increased in cells of three lines as their protein synthesis was inhibited. In summary, hematopoietic cells of different lineages and stages of differentiation constitutively express RAR-alpha mRNA. This expression is unaffected either by terminal differentiation or cell cycle. The RAR-alpha mRNA is short-lived and super-inducible by a protein synthesis inhibitor.     

Evidence for a labile intermediate in the butyrate induced reduction of the level of c-myc RNA in SW837 rectal carcinoma cells

We have found that the differentiation inducer butyric acid causes the synthesis of a cellular protein(s) that mediates a rapid decline in the level of myc RNA in SW837, a cell line derived from a human adenocarcinoma of the rectum. This effect was dose-dependent and was maximal at 1 mM. Among the short chain fatty acids tested, butyric acid was found to be the most potent. Valeric acid was less effective, and acetic, propionic, isobutyric, and caproic acids did not cause a significant change in myc RNA level. Dimethylsulfoxide, another inducer of differentiation, also caused a marked diminution of myc RNA level, but was only tested at a relatively high dose (282 mM). The reduction in myc RNA level caused by butyrate was blocked by inhibitors of protein synthesis, and was rapidly reversed by removing the inducer. This suggests that butyrate causes the induction of a labile activity that has a negative effect on myc RNA abundance.     

Common acute lymphoblastic leukemia characterized by four different DNA stemlines with heterogeneity in RNA content, antigen expression and sensitivity to chemotherapy

Four subpopulations of cells with different DNA content were present in the bone marrow of a pediatric patient with acute lymphoblastic leukemia. Flow cytometry of DNA/RNA and DNA/surface antigen expression enabled the identification and characterization of diploid, hypertriploid, tetraploid and hypertetraploid leukemic cells. This was not appreciated by cytogenetic analysis, which identified only some tetraploid cells (3/20 metaphases). Common acute lymphoblastic leukemia antigen was expressed in all aneuploid and also on 17% of diploid cells. Quantitative CALLA expression was unrelated to ploidy and cell size. Cellular RNA content paralleled ploidy, i.e. the more aneuploid cells had increased RNA content and there was pronounced RNA heterogeneity within each DNA stemline. The different subclones showed almost identical stages of early B-cell differentiation. The early pre B-cell antigens BL1 and BL2 were expressed in approx. 80 and 60%, respectively, of aneuploid leukemic cells. Cytoplasmic immunoglobulin heavy chain was also present. Clonal excess of lambda light chain immunoglobulin on the cell surface was present on less than 10% of hypertriploid, tetraploid and hypertetraploid leukemic cells indicating differentiation of a subpopulation of aneuploid leukemic cells to mature B cells. This was not seen for any diploid cells. The heterogeneity of the different subpopulations was also evident in the differences in response to chemotherapy: the hypertriploid and hypertetraploid subpopulations were most sensitive to initial induction chemotherapy.