Cytomorphological changes in human tumor cells exposed to high-polar compounds

The effects of some high-polar compounds on the cytomorphological characteristics of cultured cells of synovial and osteogenic human sarcomas (Sa-2 and Sa-4) were studied. Incubation of Sa-2 cells with 1 or 2% dimethylsulfoxide (DMSO) or 100 mM dimethylformamide (DMF) during 4-6 days induced the cellular, structural and functional changes in Sa-2 cells which were considered as manifestations of pseudoepithelial differentiation. DMSO did not influence the activity of Sa-2 cell enzymes, while DMF suppressed their activity, mainly that of acid and alkaline phosphatases. DMSO and DMF induced in Sa-4 cultured the appearance of the cells which were similar cytomorphologically to normal osteoblasts. DMSO and DMF significantly depressed the activity of acid phosphatase in Sa-4 cells and transformed the positive reaction of alkaline phosphatase to the negative one. DMSO and DMF prolonged the time of the doubling of Sa-2 and Sa-4 cells. The effects of DMSO and DMF were reversible. Methylformamide (200 mM) and dimethyl acetamide (0,1 and 10 mM) did not induce analogous changes in human sarcoma cells.     

In Vitro Phenotypic Alteration of Human Melanoma Cells Induced by Differentiating Agents: Heterogeneous Effects on Cellular Growth and Morphology,Enzymatic Activity,and Antigenic Expression

Sodium butyrate (butyrate), 5-azacytidine (5Aza-C), dimethyl sulfoxide (DMSO), and dimethyl formamide (DMF) were applied to a human melanoma cell line for the purpose of inducing pigmentation and terminal differentiation. The results are summarized as follows: 1) butyrate, DMSO, and DMF had a strong cytostatic effect, arresting cells in the G₁ phase of the cycle; 2) butyrate caused a morphological change to spindle shape whereas DMSO and DMF produced rounded cells, without affecting the levels of vimentin and intermediate filaments; 3) tyrosinase activity and melanization were stimulated by DMSO and DMF but not by butyrate; 4) butyrate induced several membrane-bound enzyme activities (alkaline phosphatase and -γ-glutamyl transpeptidase); 5) changes in the expression of antigens related to tyrosinase activity (2B7 and 5C12) only partly corresponded to the changes in enzyme activity; 6) expression of the melanosomal B863 antigen was decreased by butyrate, DMSO, and DMF; and 7) the action of DMF resembled that of DMSO whereas 5Aza-C had little effect. The results indicate that these differentiating agents activate different sets of genes, the melanogenic pathway being activated independently of -γ-glutamyltranspeptidase. The down regulation of B8G3 antigen by these agents may provide a common focus for understanding the essential action of differentiation inducers in melanoma cells.     

In vitro phenotypic alteration of human melanoma cells induced by differentiating agents: heterogeneous effects on cellular growth and morphology, enzymatic activity, and antigenic expression.

Sodium butyrate (butyrate), 5-azacytidine (5Aza-C), dimethyl sulfoxide (DMSO), and dimethyl formamide (DMF) were applied to a human melanoma cell line for the purpose of inducing pigmentation and terminal differentiation. The results are summarized as follows: 1) butyrate, DMSO, and DMF had a strong cytostatic effect, arresting cells in the G1 phase of the cycle; 2) butyrate caused a morphological change to spindle shape whereas DMSO and DMF produced rounded cells, without affecting the levels of vimentin and intermediate filaments; 3) tyrosinase activity and melanization were stimulated by DMSO and DMF but not by butyrate; 4) butyrate induced several membrane-bound enzyme activities (alkaline phosphatase and gamma-glutamyl transpeptidase); 5) changes in the expression of antigens related to tyrosinase activity (2B7 and 5C12) only partly corresponded to the changes in enzyme activity; 6) expression of the melanosomal B8G3 antigen was decreased by butyrate, DMSO, and DMF; and 7) the action of DMF resembled that of DMSO whereas 5Aza-C had little effect. The results indicate that these differentiating agents activate different sets of genes, the melanogenic pathway being activated independently of gamma-glutamyltranspeptidase. The down regulation of B8G3 antigen by these agents may provide a common focus for understanding the essential action of differentiation inducers in melanoma cells.     

Effect of dimethylsulfoxide on the morphology and proliferation of the tumor NGUK-1 cell line

The effects of low doses of dimethyl sulfoxide (DMSO) on the growth and morphology of tumour NGUK-1 strain cells from neurinoma were studied. DMSO produced a dose-dependent reduction in the proliferative capacity. 3% and 5% DMSO concentration inhibited the mitotic activity in the culture and the entry of cells into S-phase of the cell cycle, which was demonstrated by decreased mitotic and thymidine-labelling index. Electron microscopic studies at these DMSO concentrations have revealed large cisterns of rough endoplasmic reticulum with electron dense fine granules. Nucleoli had a spongy structure. DMSO induced stimulation of protein synthesis in cells. At greater DMSO concentrations almost all the cells died. At a 1% concentration DMSO had no effect on cellular morphology and proliferation of in vitro propagated tumour cells.     

In vivo and in vitro oxidative biotransformation of dimethylformamide in rat

In rats and in humans, dimethylformamide (DMF) is mainly metabolized into N-hydroxymethyl-N-methylformamide (DMF-OH). The in vitro oxidation of DMF by rat liver microsomes is decreased in the presence of catalase and superoxide dismutase. The radical scavengers, dimethylsulfoxide (DMSO), tertiary butyl alcohol (t-butanol), aminopyrine, hydroquinone and trichloroacetonitrile reduce the oxidation of DMF to DMF-OH in vitro and in vivo. Conversely, DMF inhibits the demethylation of DMSO, t-butanol and aminopyrine. The addition of iron-EDTA to the incubation system induces the production of N-methylformamide (NMF) from DMF. These results support the hypothesis that the metabolic pathway leading from DMF to DMF-OH and NMF involves hydroxyl radicals. Superoxide radical and hydrogen peroxide take part in the metabolic process. DMF is preferentially metabolized into DMF-OH. NMF appears mainly when the production of hydroxyl radicals is stimulated, the methyl group being recovered as formic acid.     

Hemoglobin synthesis in murine virus-induced leukemic cells in vitro. 3. Effects of 5-bromo-2'-deoxyuridine, dimethylformamide and dimethylsulfoxide

Erythroid differentiation of Friend leukemia cells is enhanced when the cells are grown for four days in the presence of dimethylsulfoxide (DMSO). Dimethylformamide (DMF) has a similar though less marked effect. 5-Bromo-2′-deoxyuridine (BUdR) (10^?5M) inhibits both DMF- and DMSO-stimulated differentiation. For maximum inhibition, BUdR must be present during the first two days of growth, during which time DNA synthesis is maximal. The addition of BUdR after the third day has no effect. Since BUdR is incorporated into DNA and thymidine prevents BUdR inhibition of DMSO-stimulated differentiation, it is likely that BUdR acts by virtue of its incorporation into DNA. Although BUdR alone had little effect upon cell multiplication, in combination with DMSO, cell growth was inhibited up to 40%. Since the BUdR-inhibition of the DMSO effect was approximately 70%, it is unlikely that its effect on differentiation is due to selective killing of those cells which are stimulated to differentiate.     

Mitochondrial morphology and function impaired by dimethyl sulfoxide and dimethyl Formamide

In this work, the effects of two non-ionic, non-hydroxyl organic solvents, dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) on the morphology and function of isolated rat hepatic mitochondria were investigated and compared. Mitochondrial ultrastructures impaired by DMSO and DMF were clearly observed by transmission electron microscopy. Spectroscopic and polarographic results demonstrated that organic solvents induced mitochondrial swelling, enhanced the permeation to H⁺/K⁺, collapsed the potential inner mitochondrial membrane (IMM), and increased the IMM fluidity. Moreover, with organic solvents addition, the outer mitochondrial membrane (OMM) was broken, accompanied with the release of Cytochrome c, which could activate cell apoptosis signaling pathway. The role of DMSO and DMF in enhancing permeation or transient water pore formation in the mitochondrial phospholipid bilayer might be the main reason for the mitochondrial morphology and function impaired. Mitochondrial dysfunctions induced by the two organic solvents were dose-dependent, but the extents varied. Ethanol (EtOH) showed the highest potential damage on the mitochondrial morphology and functions, followed by DMF and DMSO.     

Inhibition of dimethyl sulfoxide-induced Friend erythroleukemia cell differentiation in vitro by lithium chloride

This report describes the ability of ultra-pure lithium chloride (LiCl) to influence the growth kinetics and differentiation of Friend erythroleukemia cells in vitro. LiCl (0.2-50 mEq/l) was effective in reducing the ability of Friend cells to grow in liquid suspension culture (p less than or equal to 0.001). In addition, the capacity of these erythroleukemic cells to respond to the inducing agent dimethyl sulfoxide (DMSO) was also significantly reduced (p less than or equal to 0.001). These results demonstrate that LiCl can influence not only the proliferation of erythroleukemia cells but also their subsequent differentiation after exposure to such chemical inducers.     

Inhibited morphological terminal differentiation and enhanced proliferation of cultured mouse epidermal cells at different concentrations of dimethyl sulphoxide

Dimethyl sulphoxide (DMSO), at concentrations of 1-2%, induces terminal differentiation in several different cell types in vitro and enhances the growth of newborn mouse epidermal cells in primary culture under conditions that also permit terminal differentiation. We have found that DMSO concentrations approaching 4% reversibly inhibited (with little overt toxicity) terminal differentiation of normal epidermal cells from newborn SENCAR mice. Cells cultured in medium containing 4% DMSO and calcium in excess of 1 mM did not stratify extensively or slough large amounts of keratinized debris into the medium as occurred in control cultures, nor did they form large numbers of squamous cells or keratin bundles, as revealed by light and electron microscopy. The number of detergent-insoluble cornified envelopes was similarly reduced. Long-term growth of epidermal colonies in secondary culture was optimum in 1% DMSO, this concentration also permitting normal terminal differentiation of these cells. Since DMSO had these effects on epidermal cells in vitro, it may also affect epidermal cell proliferation and terminal differentiation in vivo, an important consideration should DMSO ever be approved for topical use in the US.     

Induction of terminal differentiation and nuclear appendage(s) formation in a human myeloid leukaemia cell line (HL-60)

In vitro terminal differentiation in a female myeloid leukaemia cell line (HL-60) was induced by either of the two inducing agents, dimethylsulphoxide (DMSO) and dimethylformamide (DMF). A higher frequency of more mature myeloid cells was noted with increasing concentrations of the inducing agents up to the optimal dose limits for cell viability, and with longer post-induction incubation periods. The highest percentage of polymorphs was obtained at 8 days post-induction with 1.25% DMSO and after 6 and 8 days exposure to 90 mM DMF. A proportion of polymorphs showed non-sex specific drumstick-like nuclear appendages, which were morphologically similar to the sex-specific drumsticks found in polymorphs from normal females in vivo. The correlation between the nuclear lobe counts and the frequencies of drumstick-like appendages in polymorphs was also similar to that reported for drumsticks in blood cells in vivo. The various stages of terminal differentiation and nuclear appendage formation in polymorphs under induced differentiation were similar to those occurring in vivo. Chromosomal analyses of this cell line indicated that individual cells had lost one X chromosome, and no portion of the missing X was detected in any of the rearranged chromosomes. Since no truly sex-specific drumsticks appeared to be present in the polymorphs of this cell line containing only one X chromosome, the study supports the accepted notion that there is a correlation between drumstick frequency and the presence of one versus two X chromosomes.     

Effects of N,N-dimethylformamide and extracellular matrix on transforming growth factor-beta binding to a human colon carcinoma cell line

Alterations in the binding of transforming growth factor-beta (TGF-beta) to the MOSER human colon carcinoma cell line caused by N,N-dimethylformamide (DMF) or extracellular matrix (ECM) were examined. DMF induced a more differentiated phenotype in the MOSER cells and resulted in a twofold increase in TGF-beta binding to the cells. This was due to an increase in receptor number with no significant alteration in the KD. The extent of increased TGF-beta binding was dependent on the dose and time of exposure to DMF. Upon removal of DMF, the receptor level returned to that of untreated cells within 6 hr. The binding of TGF-beta 1 and TGF-beta 2 to the cells was increased equally. Despite this increase in TGF-beta binding in the presence of DMF, the sensitivity of the MOSER cells to the growth inhibitory effects of TGF-beta was unaltered. Growth of the MOSER cells on ECM derived from a well-differentiated colon cell line increased the TGF-beta receptor number twofold without altering the KD. No change was observed if the MOSER cells were grown on ECM derived from a poorly differentiated cell line. While no alteration in sensitivity to TGF-beta was observed on cells grown in the presence of DMF, MOSER cells grown on the ECM derived from well-differentiated colon carcinoma cell lines were twofold more sensitive to the growth inhibitory effects of TGF-beta. These results indicated that growth conditions which resulted in a more differentiated phenotype resulted in an increase in the cellular receptors for TGF-beta.     

Structure of lysozyme dissolved in neat organic solvents as assessed by NMR and CD spectroscopies

The structure of the model protein hen egg-white lysozyme dissolved in water and in five neat organic solvents (ethylene glycol, methanol, dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The NMR spectra of lysozyme reveal the lack of a defined tertiary structure in all five organic solvents, although the examination of line widths suggests the possibility of some ordered structure in ethylene glycol and in methanol. The near-UV CD spectra of the protein suggest no tertiary structure in lysozyme dissolved in DMSO, formamide, and DMF, while a distinctive (albeit less pronounced than in water) tertiary structure is seen in ethylene glycol and a drastically changed one in methanol. A highly developed secondary structure was observed by far-UV CD in ethylene glycol and methanol; interestingly, the alpha-helix content of the protein in both was greater than in water, while the beta-structure content was lower. (Solvent absorbance in the far-UV region prevents conclusions about the secondary structure in DMSO, formamide and DMF.) Copyright 1999 John Wiley & Sons, Inc.     

Effects of cyclic AMP on the growth of differentiating and undifferentiated Friend erythroleukemic cells.

Elevated concentrations of cyclic AMP elicit only minor reductions in growth rate and saturation density in undifferentiated Friend erythroleukemic cells. During the course of dimethylsulfoxide (DMSO)-induced differentiation, Friend cells convert from a cyclic AMP-tolerant state to a phenotype characterized by a high degree of sensitivity to cyclic AMP-mediated growth arrest. Conversion to cyclic AMP sensitivity is detectable after 30 hours growth in medium containing 2% DMSO, and either 0.5 mM 8-Br-cyclic AMP or 5 nM cholera toxin. Cultures of differentiating Friend cells achieved a stationary phase density that was approximately 8-fold higher than the cell density observed in parallel, differentiating cultures treated with 0.5 mM 8-Br-cyclic AMP. Temporally, the appearance of cyclic AMP-sensitivity corresponds to the early expression of in vitro erythroid differentiation (Ross et al., '74), but growth arrest does not alter the subsequent accumulation of hemoglobin in non-dividing DMSO-induced cells. Since growth arrest is preceded by a round of cell division, these observations are consistent with the concept that DMSO must be present during DNA replication for the subsequent expression of hemoglobin synthesis (McClintock and Papaconstantinou, '74; Levy et al., '75; Harrison, '76).     

The effect of Solcoseryl on in-vitro cultured cells

Explants of peripherical nervous system (PNS), skin and ventriculus cordis from chick embryo were cultivated in Maximow chambers and the effect of Solcoseryl, Fa. Solco Basel AG, on some morphological parameters was tested. 1. The growth of tissue cultures is influenced by Solcoseryl in relation to concentration and time of application. The index of area in cultures of PNS and cor increased within the first days. By long time application up to 6 days in vitro the index of area decreased and the index was the same than in controls. Explants of skin showed no essential stimulation of growth. 2. The number of cells per unit of culture in the outgrowth of PNS, cor and skin was different influenced. The density of cells in cultures of PNS and skin decreased (signif. difference). In explants of heart we could not observe a difference between the inside and outside of the outgrowth. An influence of Solcoseryl on the degree of migration is discussed. 3. The area of cell nuclei from heartcells was observed. The area decreased under the influence of Solcoseryl. The difference is significant. 4. The mitotic index of heart cells increased by application of Solcoseryl within the first 2 and 3 days in vitro. 5. The number of nucleoli per nucleus of heart cells under experimental conditions increased significant. It is discussed, Solcoseryl influenced in vitro metabolic processes in suitable systems; stimulation of cell proliferation and migration and rns-synthesis was observed within the first days of cultivation. In-vitro-systems are important objects and they are suitable for tests of pharmaca in vitro.     

INDUCTION OF ERYTHROID MATURATION BY DIMETHYL SULFOXIDE IN FRIEND LEUKEMIC CELLS

Enhancement of the erythroid maturation in Friend virus-induced leukemic cells has been examined in vitro by the treatment with dimethyl sulfoxide (DMSO). Although the cell growth was inhibited in the medium containing 2% DMSO, many cells remained viable for a week. By the 3rd day of the culture, the cells treated with DMSO became more strongly agglutinated by phytohemagglutinin than the cells incubated without DMSO. Mouse erythrocyte membrane-specific antigens were also detectable at the 4th day. At the 8th day of the culture hemoglobin synthesis was apparently demonstrated in the cells treated with DMSO, which could not be seen in the untreated cells. Maturation or differentiation along the erythroid pathway in Friend leukemic cells by DMSO is discussed on these markers.     

Membrane permeabilization of phosphatidylcholine liposomes induced by cryopreservation and vitrification solutions

Membranes are the primary site of freezing injury during cryopreservation or vitrification of cells. Addition of cryoprotective agents (CPAs) can reduce freezing damage, but can also disturb membrane integrity causing leakage of intracellular constituents. The aim of this study was to investigate lipid-CPA interactions in a liposome model system to obtain insights in mechanisms of cellular protection and toxicity during cryopreservation or vitrification processing. Various CPAs were studied including dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), dimethyl formamide (DMF), and propylene glycol (PG). Protection against leakage of phosphatidylcholine liposomes encapsulated with carboxyfluorescein (CF) was studied upon CPA addition as well as after freezing-and-thawing. Molecular interactions between CPAs and phospholipid acyl chains and headgroups as well as membrane phase behavior were studied using Fourier transform infrared spectroscopy. A clear difference was observed between the effects of DMSO on PC-liposomes compared to the other CPAs tested, both for measurements on CF-retention and membrane phase behavior. All CPAs were found to inhibit membrane leakiness during freezing. However, exposure to high CPA concentrations already caused leakage before freezing, increasing in the order DMSO, EG, DMF/PG, and GLY. With DMSO, liposomes were able to withstand up to 6 M concentrations compared to only 1 M for GLY. Cholesterol addition to PC-liposomes increased membrane stability towards leakiness. DMSO was found to dehydrate the phospholipid headgroups while raising the membrane phase transition temperature, whereas the other CPAs caused an increase in the hydration level of the lipid headgroups while decreasing the membrane phase transition temperature.     

Unfractionated human marrow cell cryopreservation using dimethylsulfoxide and hydroxyethyl starch

Unfractionated bone marrow (BM) cells were cryopreserved in 1- to 2-ml aliquots using a mixture containing both 5% dimethylsulfoxide (DMSO) and 6% hydroxyethyl starch (HES) in an attempt to increase the viable cell yield and reduce the clumping after thawing, observed when 10% DMSO is used alone. Samples thawed after storage for 6 months in the vapor phase of liquid nitrogen, were assayed. Compared to prefreeze values, there was both a greater number of cells that excluded Trypan Blue (50 +/- 12 vs 28 +/- 12%, P less than .01) and a greater CFU-C Recovery (110 +/- 20 vs 89 +/- 35%, P less than .02) for cells in the DMSO/HES mixture, compared to those in 10% DMSO alone. No macroscopic clumping of the thawed cells was observed for those cryopreserved in the mixture in contrast to those in DMSO alone. Freezing was done without a rate-controlled freezing apparatus by simply placing the samples initially into a -80 degrees C freezer, and then later into a liquid nitrogen freezer. Additional samples stored in the DMSO/HES mixture were kept at only -80 degrees C, and when thawed 12 to 16 months later also gave an excellent CFU-C recovery (105 +/- 39% of prefreeze). The DMSO/HES mixture allows for a simplified BM cryopreservation technique that not only assures excellent recovery of CFU-Cs and eliminates clumping upon thawing, but also does not require either the use of a rate-controlled freezer or liquid nitrogen temperatures for storage up to a year.     

Nuclear magnetic resonance investigation of the state of water in protein solution.

The line width of the NMR signal of water protons in solutions of native actomyosin and actomyosin denatured by heat, acetone or urea was measured over the temperature range from -10 degrees to below the freezing point. The line widths of the water band which increased exponentially with decreasing temperature were compared with each other and also with those of the corresponding control solution without actomyosin. The line broadening observed for native actomyosin solution on lowering the temperature was significantly smaller than that for heat-denatured actomyosin solution. This difference implies that this signal is sensitive to conformational perturbations of the protein. In addition, the temperature dependence of the line width for heat-, acetone-, or urea-denatured actomyosin solution was similar to that for the corresponding control solution. These phenomena can be interpreted in terms of the state of water associated with the hydrophobic and hydrophilic residues. Similar NMR studies of actomyosin solution containing dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) showed that DMSO and DMF prevent the formation of ice crystals until about -70 degrees, suggesting that the cryoprotective effects of DMSO and DMF are due to the change in the state of water described above. These differences in temperature dependence between the sample and control solutions are well-correlated with the viscosity of the solution. This correlation is useful for elucidation of the mechanism of the protein denaturation.     

二甲基亚砜诱导人食管癌细胞分化的实验研究

The fact that treatment of leukemia (Acute Promyelocytic Leukemia) with ATRA (All-Trans Retinoic Acid) was so succeeded that it was considered as a good example for tumor therapy. In the treatment of solid tumors by means of induced differentiation, however, has not been yet so broken-through. DMSO (Dimethylsulfoxide) was a common and simple organic compound, which comprised a variety of biological activities. For example, DMSO induced differentiation of leukemia in many reports. However, the effect of DMSO on solid tumors was to be explored further. In the present study, DMSO was used to human esophageal cancer cell lines in vitro in comparison with the classical inducer ATRA. From the view of morphology, cell cycle, growth inhibition, cytokeratin 4 expression, dye transfer and tumorigenecity, the results demonstrated that DMSO as well as ATRA could induce differentiation of human esophageal cancer cells. Interestingly, DMSO was confirmed to be more effective in inducing differentiation of esophageal cancer cells than ATRA. It suggests that DMSO showed some good prospects for the treatment of solid tumors.     

二甲基亚砜诱导人食管癌细胞分化的实验研究

维甲酸诱导分化治疗白血病取得巨大成功,为治疗肿瘤开辟了新思路,但诱导分化治疗实体瘤目前尚无突破性进展。二甲基亚砜有多种生物学作用如诱导白血病细胞分化,对实体瘤细胞的作用研究却不多。本文将二甲基亚砜试用于体外培养的人食管癌细胞株,同时设维甲酸作参照,从形态和细胞周期改变、角蛋白表达及成瘤性等多方面证实二甲基亚砜和维甲酸一样可诱导食管癌细胞分化,并且发现二甲基亚砜的作用优于维甲酸,强烈提示二甲基亚砜对实体瘤治疗的可能应用前景。