DMSO-treated Cultures Research Articles

Pomalidomide Modulates Transcription Networks Regulating Human Erythropoiesis and Globin Switching: Implications for Treatment of Hemoglobinopathies

Sickle cell disease (SCD) represents a major challenge in hematology, with approximately 100,000 Americans afflicted and the annual number of newborns with SCD set to rise over the next 40 years worldwide. Current treatment approaches rely on increasing levels of fetal hemoglobin (HbF) to prevent painful vaso-occlusive crises and hemolysis secondary to red cell sickling. Hydroxyurea remains the only pharmacologic intervention approved for SCD; however, it has limited efficacy and carries significant side effects such as myelosuppression. Thus, there is a critical need to develop drugs that enhance HbF production without similar dose limiting side effects. Second generation immunomodulatory drugs, such as pomalidomide, are a class of emerging HbF inducers both in vitro and in vivo. Recent work from our laboratory revealed that hydroxyurea and pomalidomide differentially regulate HbF production in CD34+ cells undergoing erythroid differentiation using a 3-phase culture system. Pomalidomide, but not hydroxyurea, was found to decrease BCL11A expression through a yet to be defined mechanism. In the present study, we sought to characterize erythropoiesis and the expression of key transcription factor networks in this 3-phase culture system to determine the mechanisms underlying pomalidomide’s effect. Following a four day expansion period, isolated CD34+ cells from the peripheral blood of SCD or normal individuals were differentiated along erythroid lineage in the presence of pomalidomide (1μM) or DMSO (vehicle) for 14 days. As an additional control, CD34+ cells were also treated with hydroxyurea (10μM). Proliferation and erythroid differentiation were assessed at 7, 11 and 14 days of culture. Although a 50% decrease in cell growth was noted in cells treated with hydroxyurea, no such decrement was found in control, DMSO and pomalidomide-treated cells. Moreover, pomalidomide produced a transient delay in erythroid differentiation between days 6 and 11 of culture phenotypically documented by flow cytometric analysis using glycophorin-A, α-4 integrin and band 3 as surface markers monitoring erythroid differentiation as well as morphologically by May-Grunwald Giemsa staining. In contrast, cells treated with hydroxyurea demonstrated accelerated differentiation, compared to the control cultures. However, by day 14 of culture, no significant difference was observed under any condition, suggesting that the delayed cells eventually finished terminal differentiation. In terms of HbF induction, we confirmed elevated production in the cultures with pomalidomide by measuring the number of F-cells by flow cytometry. We also evaluated the production of γ-globin chains by qRT-PCR and western blot at D4 and D11 and found a dramatic increase in the production of γ-globin, in both SCD and normal samples treated with pomalidomide.We posited that pomalidomide might foster changes in transcription factors known to play a role in both erythropoiesis and globin switching. To this end, we evaluated the expression kinetics of BCL11A, SOX6, KLF1, MI2β, GATA1 and FOG1 via qRT-PCR and western blot analyses. In DMSO-treated cultures the above transcription factors were maximally expressed between days 6-8, and their levels diminished during the remainder of the culture. Conversely, pomalidomide markedly decreased BCL11A, SOX6, KLF1 and MI2β between days 4 and 6 in cultures of both SCD and normal samples. In line with our results, MI2β acts as a positive regulator of BCL11A and KLF1, and previous studies have shown that its knock down in CD34 cells leads to decreased levels of BCL11A and KLF1. Western blot analyses confirmed the qRT-PCR data. Further, the divergent expression patterns correlated temporally with the differentiation delay suggesting that pomalidomide modulates expression of members of the BCL11Atranscription factor complex, thereby augmenting γ-globin production.Taken together, these data provide evidence that pomalidomide influences erythropoiesis by modulating transcription factor expression in CD34+ cells differentiated in the 3-phase culture system, leading to a decrease in BCL11A and activation of γ-globin production. Importantly, further exploration of these pathways that function to regulate erythropoiesis and promote HbF silencing, may help elucidate the mechanism of action of pomalidomide as well as identify additional druggable molecules. DisclosuresNo relevant conflicts of interest to declare.

Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1

Specific deletion of Notch1 and RBPjkappa in the mouse results in abrogation of definitive haematopoiesis concomitant with the loss of arterial identity at embryonic stage. As prior arterial determination is likely to be required for the generation of embryonic haematopoiesis, it is difficult to establish the specific haematopoietic role of Notch in these mutants. By analysing different Notch-ligand-null embryos, we now show that Jagged1 is not required for the establishment of the arterial fate but it is required for the correct execution of the definitive haematopoietic programme, including expression of GATA2 in the dorsal aorta. Moreover, successful haematopoietic rescue of the Jagged1-null AGM cells was obtained by culturing them with Jagged1-expressing stromal cells or by lentiviral-mediated transduction of the GATA2 gene. Taken together, our results indicate that Jagged1-mediated activation of Notch1 is responsible for regulating GATA2 expression in the AGM, which in turn is essential for definitive haematopoiesis in the mouse.

Cytogenetic evaluation of butylated hydroxytoluene

The genotoxicity of butylated hydroxytoluene (BHT) was evaluated using different cytogenetic short-term tests. Chromosome aberrations and anaphase-telophase alterations were analyzed in Chinese hamster ovary (CHO) cells. The induction of sister chromatid exchanges (SCEs) was evaluated in CHO cells as well as in human peripheral blood lymphocytes treated during all the culture time or for the last 24 h of incubation. The cell cycle progression was determined in cultures with BrdU added. Doses of 0.10, 0.25 and 0.50 μg/ml of the compound diluted in DMSO were employed. Untreated and DMSO-treated cultures were used as controls. Results obtained showed that BHT induced a significant increase of chromatid and chromosome breaks with the three doses employed, but did not increase the SCE frequencies either in CHO cells or in human lymphocytes under the experimental conditions employed. In anaphase-telophase, the compound induced a significant increase of multipolar mitoses. On the other hand, cytotoxicity of BHT was evidenced by the lack of cells in second mitosis with the highest dose and the decrease of the mitotic index in CHO cells as well as by the delay of the cell cycle progression both in CHO cells and human lymphocytes. Despite this, chromosomal damage was only induced by doses raising the cytotoxic level. The results obtained can be considered as evidence of BHT genotoxicity.

Proteoglycan synthesis in human erythroleukaemia (HEL) cells.

Synthesis of sulphated proteoglycans was compared in human erythroleukaemia (HEL) cells grown under control conditions and under stimulation by dimethyl sulphoxide (DMSO) and phorbol 12-myristate 13-acetate (PMA). Synthesis of [35S]sulphate-labelled proteoglycans by DMSO-treated cells was decreased by about 35% relative to controls, but synthesis of proteoglycans by PMA-treated cells increased 3-4-fold. Control and DMSO-treated cells secreted 65% of the newly synthesized proteoglycans, but PMA-treated cells secreted more than 90%. Sepharose CL-6B chromatography and SDS/PAGE suggested the presence of several proteoglycans in the cells and culture medium. The PMA-treated cells synthesized a low-Mr proteoglycan (Kav. 0.3( that was not present in controls and DMSO-treated cultures. The proteoglycans of the cells and medium from control, DMSO-treated and PMA-treated cultures could be separated into three fractions by octyl-Sepharose chromatography. The proteoglycans were resistant to trypsin but were degraded by Pronase and papain to fragments similar in size to the NaOH/NaBH4-generated glycosaminoglycans. The average chain length of the glycosaminoglycans (Kav. 0.20 on Sepharose CL-6B for controls) was decreased by DMSO (Kav. 0.25) and by PMA (Kav. 0.30-0.38). Chondroitin ABC lyase digestion of the proteoglycans from the medium of the control cultures produced two core proteins at Mr 31,000 and 36,000. The DMSO medium proteoglycans had only the 31,000-Mr core protein, and the PMA culture medium proteoglycans had core proteins of Mr 27,000, 31,000 and 36,000. Changes in synthesis of proteoglycans induced by DMSO or PMA may have relevance for the maturation of haematopoietic cells.

Developmental effect of dimethyl sulfoxide on hypothalamo-neurohypophysial neurons in vitro.

Primary dissociated cultures were established from diencephalic tissue of 14-day-old fetal rats. Neurons exhibiting immunocytochemical staining for neurophysin appeared in these cultures after 6 days of cultivation. Addition of dimethyl sulfoxide (DMSO) to the culture medium resulted in a slight decrease in total neuronal cell mass as assessed by immunocytochemistry and radio-immunometric quantitation of neuron-specific enolase. In contrast, in DMSO-treated cultures the number of neurophysin-immunoreactive neurons was more than doubled as compared to control cultures. [3H]Thymidine labeling and autoradiography in conjunction with immunocytochemistry for neurophysin showed that this was not due to a mitogenic effect of DMSO on precursor cells. Time-course analysis of the action of DMSO revealed a 6-day time lag between the initiation of treatment and the appearance of increased numbers of neurophysin-immunoreactive cells. These findings suggest that DMSO, which has previously been reported to have a differentiation-inducing effect on malignant transformed cells, may also modulate cellular processes that control differentiation in specific types of neurons in primary culture.

Characterization of the growth inhibited substate induced in murine hepatic tumor cells during in vitro exposure to dimethylsulfoxide

Kinetic events associated with the dimethylsulfoxide (DMSO)-induced inhibition of hepatic tumor cell proliferation were studied using established lines of murine liver tumor cells (BW77-2 and Hepa-1/A1) and conditions of polar solvent treatment (1-3% final concentration in the culture medium for a period of 4 days) previously shown to increase the expression of differentiated functions in BW77-2 cells. Cell-cycle substrates of exponentially growing and DMSO-treated liver tumor cell populations were compared by flow cytometric techniques employing recently developed cytochemical criteria to identify hepatocyte cell cycle compartments based on individual cellular RNA and DNA contents (Higgins, 1985). Suppression of hepatic tumor cell proliferation by DMSO (in non-cytotoxic concentrations) persisted only for the duration of the exposure period. Resumption of cell division was readily observed following removal of the polar solvent from the culture medium. During DMSO treatment, BW77-2 and Hepa-1/A1 cells accumulated in the G1 phase of the cell division cycle (low-population-density 3% DMSO-treated cultures were composed of 88% G1 cells compared to only 48% G1 DNA content cells in control cultures of similar population density) and exhibited a substantial shift to lower mean cellular RNA content. The relatively few S- and G2 + M-phase cells in DMSO cultures also possessed lower RNA contents compared to the corresponding cell cycle compartments in exponentially growing cultures. The mean RNA contents for the G1, S, and G2 + M compartments of DMSO-treated cells approximated 63.8, 78.6, and 74.4%, respectively, of the amounts observed in control cultures. Low-RNA G1 cells in DMSO cultures expressed a continuum of RNA distributions similar in range variation to (but at lower mean cellular RNA content levels than) cycling G1 cells in log-phase growth. Thus, G1 cells in 1% DMSO-treated populations had a mean cellular RNA content of just 25 (arbitrary RNA) units compared to over 40 units for G1 cells in exponential phase growth. Low RNA content, non-replicating, hepatic tumor cells in polar solvent-treated cultures were designated as being in the "Qi" substate (DMSO-induced quiescent-type cells). Release of BW77-2 cells from Qi, after replacement of the DMSO-containing growth medium by medium without the polar solvent, was characterized by an increase in mean G1 RNA content and recruitment into log-phase growth.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of Retinoic Acid Versus Dimethyl Sulfoxide on Friend Erythroleukemia Cell Growth. I. Cell Proliferation, RNA Content, and Protein Content<xref ref-type="fn" rid="FN2">2</xref>

The effect of all-trans-retinoic acid (RA), an oxidative product of vitamin A, on cell growth, cell cycle kinetics, RNA content, and protein content of exponentially growing Friend erythroleukemia (FL) cells was determined and compared with the results obtained with dimethyl sulfoxide [(DMSO) CAS: 67-68-5; methyl sulfoxide], an inducer of differentiation, and alpha-difluoromethylornithine (DFMO), a potent inhibitor of ornithine decarboxylase (EC 4.1.1.17) activity. Growth inhibition of FL cells was observed only during continuous treatment with RA. While RA did not prevent growth to a high cell density, if cultures were maintained in exponential growth, cell number was reduced by 37, 67.4, and 72.2% after 6-day exposure to 10(-7), 10(-6), and 10(-5) M RA, respectively. In comparison, 280 mM (2%) DMSO and 5 mM DFMO inhibited growth over the same time course by 92 and 97.3%, respectively. DMSO resulted in an early, transient (18-24 hr) accumulation of cells in G1 phase followed by a later (5 day), irreversible accumulation of G1 cells. RA required several cell generations (48-72 hr) before a dose-dependent G1 accumulation was observed. Two populations of RA-treated FL cells could be identified: one with an intermediate RNA content (T-cells) similar to near-plateau-phase control cultures and the other with low RNA content (Q-cells) similar to that observed for DMSO-differentiated (D) cells. The kinetics of the decrease in RNA content of Q-cells paralleled those of D-cells in DMSO-treated cultures; the proportion of Q- versus T-cells in RA-treated cultures was dependent on both concentration and length of exposure. DFMO treatment did not give rise to low-RNA-containing Q-cells. Protein content of RA-treated cells was also diminished and approached that observed for hemoglobin-containing D-cells. FL cells were recoverable from long-term (greater than 5 days) treatment with RA, though 2-3 days were required for reestablishment of exponentially growing cultures; apparently only moderate RNA-containing T-cells repopulated the culture. Neither RA nor DFMO treatment gave rise to benzedine-positive, hemoglobin-containing cells as compared to DMSO that induced differentiation in these cultures.

Secretion of albumin and alpha-foetoprotein by dimethylsulphoxide-stimulated hepatocellular carcinoma cells

Exposure of BW77-1 and BW77-2 mouse hepatic tumour cells to the polar solvent dimethylsulphoxide (DMSO) altered extracellular accumulation of albumin and alpha-foetoprotein (AFP) and perturbed their cell cycle kinetics. The amount of albumin secreted into the culture growth medium was dependent on the concentration of DMSO used. Hepatic tumour cells cultured in 1 and 2% DMSO accumulated 50% and 111% more albumin, respectively, than non-DMSO-stimulated cells during the final 24 h of a 4-day exposure to the polar solvent. Commitment of mouse hepatoma cells to increased albumin secretion was temporally dependent, requiring a minimum of 48 h in the presence of DMSO. The AFP level in 1% DMSO-treated cultures was also significantly increased, compared with control cells. Unlike albumin secretion, however, exposure of hepatic tumour cells to 2% DMSO did not further increase (but slightly decreased) extracellular AFP accumulation. Treatment of BW77-1 cells with DMSO resulted in a gradual decline in the percentage of 2C DNA content cells (diploid G1 population) and in a corresponding increase in the proportion of cells with a 4C DNA content (generation of either a G2 or tetraploid G1 population). The extent of this shift directly reflected the concentration of polar solvent in the medium and paralleled the DMSO-induced stimulation in albumin secretion. DMSO-stimulated hepatic tumour cells, therefore, may prove useful in the elucidation of specific regulatory events underlying control of gene expression during the hepatocyte cell cycle.

Hyperosmotic injury in mammalian cells. 3. Volume and alkali cation alterations of CHO cells in unprotected and DMSO-treated cultures

Correlated studies on volume distributions and cation (Na + and K +) content of CHO cells in suspension were carried out after various exposures to hypertonic NaCl or sucrose (500–7550 m Osm in both the presence and absence of DMSO (5–20%; w v ). The effects superimposed by ouabain (10 −2–10 −4 m), amphotericin B (6–18 μg/ml), and glutaraldehyde (1.25%) on the above-mentioned parameters were also investigated. Volumetric analysis of CHO cells with the Coulter Channelyzer indicated a biphasic dose-dependent response to hypertonic media, the duration of the TABLE 2 Correlation between Volume, Survival, and Cation Content of CHO Cells Exposed to Hypertonic Media in Suspension Osmolality m Osm Exposure (min) Hypertonic agent NaCl Sucrose V a Na + K + S b V Na + K + S 1000 60 or less Small High High High Normal Low High High 1500–2000 60 or less Small High Low Low Normal Low High High 2000 or over 60 or more Small or large c High Very low Very low Small Very low Very low Very low a V, modal volume. b S, surviving fraction [data from Ref. (18)]. c Time dependency visible during a 2-hr observation period. shrinkage- re-expansion cycle being inversely proportional to medium osmolality. In hypertonic sucrose, CHO cells apparently fail to shrink up to approximately 1000 m Osm. In both hypertonic sucrose and NaCl there is a constant loss of intracellular K + at velocities which depend primarily on medium tonicity and also on exposure length. K + loss is accompanied by increased levels of intracellular Na + only in cells exposed to hypertonic salt; in hypertonic (1–2 m) sucrose intracellular Na + shows a progressive diminution. In NaCl-DMSO experiments the amount of K + lost from the cells is less in comparison to that observed in “unprotected” cell suspensions. DMSO also mitigates excessive intracellular accumulations of Na +. In contrast to ouabain and amphotericin B, DMSO was found to slow down considerably the early volume re-expansion phase. Glutaraldehyde on the other hand totally suppressed the ability of cells to readjust their volume in hypertonic solutions. By correlating the present data with several earlier published results on CHO cells' posthypertonic survival, it appears that intracellular K + plays a key role in the chain of reactions that enable nucleated mammalian cells to sustain hypertonic stresses. Whether intracellular K + is equally important for the survival of cells at subzero temperatures remains to be determined.

Enhanced parainfluenza I (6/94) virus detection in latently infected human brain cell cultures by treatment with cytochalasin D and dimethyl sulfoxide

The ability of cytochalasin D (CD) and dimethyl sulfoxide (DMSO) to enhance parainfluenza I (6/94) virus replication was studied in various cell culture systems. Treatment of CV1 cells with CD (1 microgram/ml) dissolved in DMSO prior to primary 6/94 virus exposure at 10(0)--10(5) multiplicities of infection did not substantially enhance virus replication. However, there was a transient increase in cell associated virus one day after infection of DMSO-treated cultures. CD treatment of cultures of human brain cells latently infected with 6/94 virus (LIHB cells) did not enhance 6/94 virus detection. Cocultivation of CV1 cells with CD-treated LIHB cell cultures, and cocultivation of LIHB cell cultures with CD-treated CV1 cells, resulted in the production of both cell-associated and cell-free 6/94 virus three and five days after cocultivation. No virus was detected after similar cocultivation of untreated LIHB cell cultures with untreated CV1 cells. The usefulness of CD-DMSO treatment in the rescue of virus from 6/94 LIHB cell cultures appears limited to a cocultivation system. The use of these techniques to enhance virus rescue from human tissues suspected of harboring latent viral genomes is discussed.

Hyperosmotic injury in mammalian cells 2. Surface alterations of CHO cells in unprotected and DMSO-treated cultures

Chinese hamster ovary (CHO) cells exposed for up to 2 hr to hypertonic Eagle's MEM were examined for surface changes by scanning electron microscopy. Cells hypertonically stressed in the presence of DMSO were identically monitored. In media supplemented with NaCl, little changes were observed below 2600 m osm. Above this level there is a concentration-and time-dependent disappearance of surface microvilli and blebs as well as a reduction in the plasma membrane ruffling activity. At hyperosmolalities of about 3000 mosM the cells became devoid of any surface projections and demonstrated multiple blister-like patches. In 20 × isotonic solution, the cells are flattened, the usual surface projections are absent, and the membrane appears to be full of pits and holes, giving the cells the appearance of an overcooked fried egg. In hypertonic sucrose the surface of CHO cells developed a gradual thickening and agglutination of surface components at tonicities of about 5000 m osm. DMSO at 5 or 10% (w/v) concentration considerably mitigated the surface alterations of hypertonically NaCl stressed cells. Enzymatic digestions with pronase simulated some of the changes seen with hyperosmotic salt. Neuraminidase treatment produced no demonstrable change in surface topography.

Hyperosmotic injury in mammalian cells 1. Survival of CHO cells in unprotected and DMSO-treated cultures

Cultures of CHO cells growing as monolayers or held in suspension were exposed to increased concentrations of NaCl in Eagle's MEM. Analysis of posthypertonic survival indicated that the cells' ability to withstand hyperosmolal (596–7550 mos m) environment is anchorage-dependent, cells in suspension being extremely sensitive to hyperosmotic conditions. By trypsinizing monolayers at various intervals during hypertonic exposure (3300 or 7550 mos m), the existence of a repair mechanism has been revealed. The repair is abolished by inhibitors of RNA or DNA synthesis. Media made hyperosmolal with nonpenetrating sugars (sucrose, sorbitol) do not elicit a repair process. DMSO at 5 or 10% levels is able to prevent the hypertonic killing of cells growing as monolayers. Cells in suspension which have been allowed to recover from the trypsin action, but not the freshly trypsinized cells, were also protected by DMSO. The DMSO effect seems to be mediated by the presence, probably at the membrane level, of a trypsin-removable material (glycoprotein?).