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Fibronectin (FN) is a major component of the extracellular matrix which plays important roles in a variety of cellular processes including cell adhesion, and migration. The soluble cellular form of FN has a monomer molecular weight of approximately 250 kDa, and generally exists as a dimer of 500 kDa. We have isolated a different form of soluble FN from mouse breast cancer cell line SC115 conditioned medium (CM) and purified it to homogeneity as evidenced by both native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE. It still exhibits a monomeric form of about 250 kDa while its form in the CM is stable and soluble with an apparent tetrameric molecular weight in the range of 800-1000 kDa. This form of FN is a potent cell adhesion factor (AF) that induces adhesion to polystyrene, elongation, spreading, alignment or "track" formation, and migration of mouse erythroleukemia cells. Column fractions homogeneous for AF protein were able to stimulate 10% cell adhesion at concentrations of 23 ng/ml and 1.9 ng/cm(2). Purified AF induced 50% cell adhesion at 94 ng/ml and 7.5 ng/cm(2). AF also increased the migration of human aortic smooth muscle and vascular endothelial cells. However, this form of FN differs from other forms as it does not bind tightly to either gelatin or heparin. Studies of this AF should shed light on adhesion of cells to extracellular matrix molecules and on cell migration, both of which are critical in several biological processes such as wound healing, metastasis, matrix formation and structure, and organ development.

Patients with acute promyelocytic leukemia (APL) associated with the t(15;17) translocation and fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RAR-alpha) genes achieve complete remission but not cure with all-trans retinoic acid (RA), NB4, a cell line derived from a patient with t(15;17) APL that undergoes granulocytic differentiation when treated with pharmacologic doses of RA, was used as a model for differentiation therapy of APL. We found that NB4 cells are resistant to differentiation by nonretinoid inducers such as hexamethylene bisacetamide (HMBA), butyrates, vitamin D3, or hypoxanthine, all of which can induce differentiation in the commonly used HL60 leukemia cell line. Preexposure of NB4 cells to low concentrations of RA for a period as short as 30 minutes abolished resistance to nonretinoids and potentiated differentiation. Sequential RA and HMBA treatment yielded maximal differentiation by 3 days of drug exposure, whereas the effect of RA alone peaked after 6 days and yielded a smaller percentage of differentiated cells. RA also reversed NB4 cell resistance to butyrates and allowed for synergistic differentiation by these agents. Pretreatment with HMBA before exposure to RA failed to stimulate differentiation. Sequential RA/HMBA treatment also markedly increased the extent of differentiation of primary cultures of bone marrow and peripheral blood mononuclear cells from three APL patients. In one case RA/HMBA treatment overcame resistance to RA in vitro. Together, these results suggest that intermittent low doses of RA followed by either HMBA or butyrates may be a useful combination in the treatment of APL. This clinical strategy may help prevent or overcome RA resistance in APL.

Treatment of mouse erythroleukemia (MEL) cells with hexamethylene bisacetamide induces a program of erythrodifferentiation, as judged by an increase in the synthesis of globins and other erythroid-specific products. This induction can be inhibited by glucocorticoids, e.g. dexamethasone. All globin and other erythroid-specific genes tested contain GATA response elements (GATA-RE) and can be transactivated by GATA-1, a transcription factor. GATA-1 is highly expressed in erythroid cells, including MEL cells. We noted a glucocorticoid receptor (GR) response element motif near a GATA-RE motif in the promoter region of the mouse beta-major and beta-minor globin genes and about 130 bases away from a GATA-RE in the alpha 1-globin gene promoter and, therefore, investigated the possibility that the dexamethasone-induced inhibition of induced MEL cell differentiation may involve effects of the GR on GATA-1 activity. Evidence obtained from transfection assays and DNA electrophoretic mobility shift assays indicates that the GR binds GATA-1 and interferes with its function before any interaction with DNA, but that the presence of a glucocorticoid response element near a GATA-RE augments the GR effect. The N-terminal 106-amino acid domain of the GR was found to be essential for the effect, possibly by binding to GATA-1. Since GATA-1 is autoregulatory, i.e. it has been shown by others to bind to its own promoter and up-regulate its own transcription, the finding that activated GR can interfere with GATA-1 function may provide an explanation for the inhibition by glucocorticoids of the entire program of erythroid differentiation in MEL cells. That is, by interfering with GATA-1 function, the GR inhibits not only the expression of erythroid structural genes, but may also inhibit the expression of a primary erythroid regulatory gene, GATA-1. It was also shown that the GATA-RE in each of the beta-globin promoters responds to mouse GATA-1 in a functional transfection assay.

The addition of certain proteases to cultures of Friend virus-infected mouse erythroleukemia cells can induced up to 90% of the cells in culture to become hemoglobin-containing, as assessed by positive staining for benzidine (B+). Because the mechanism of this protease action is unknown, media components were studied as possible targets for protease activity. Aliquots of medium plus serum were incubated for various times with levels of protease sufficient to induce approximately 50% of the cells to the B+ state. Cells were added to protease-pretreated serum either before or after inactivation of the protease. In all cases, enzymatically active protease had to be present with the cells to induce B+ cells to form. Serum and other components of the medium pretreated with protease were inactive. Mouse erythroleukemia cells grown in the absence of serum were also induced by proteases to form B+ cells. These data imply that the inducing action of proteases cannot be passively transferred by protease-pretreated serum or medium nor is serum required for protease-mediated induction of B+ cells. Taken together, these conclusions suggest that the protease action is on the cells or on cellular products intimately associated with cells.

Monomeric (G-) actin and filamentous (F-) actin levels were determined in Triton X-100 extracts prepared from mouse erythroid cells at various stages of differentiation. G-actin and F-actin were found in the Triton-soluble fraction and in the Triton-insoluble fraction, respectively. G-actin levels in untreated and dimethyl sulfoxide-treated (differentiated) erythroleukemia cells, reticulocytes, and erythrocytes were 48, 33, 2.8, and 0.37 microgram/mg protein, respectively, and F-actin levels were 17, 35, 45, and 59 micrograms/mg protein, respectively. G-actin/F-actin ratios were successively lower in cells representing the more mature stages of development.

The relative amounts of hemoglobin (Hb) major and Hb minor accumulated during induction of erythrodifferentiation in mouse erythroleukemia (MEL) cells were studied. The ratio of major to minor was found to depend not only upon the inducer tested (as reported previously by others), but also upon the concentration of the inducer and the time of exposure to the inducer as well as the specific cell line of MEL cells studied. At concentrations required for optimal induction of differentiation, certain agents led to the accumulation of predominantly Hb major, but suboptimal concentrations of the same inducers led to predominantly Hb minor accumulation. After a relatively short induction time (2 da) utilizing a given inducer either the level of Hb minor was higher than that of Hb major or the levels of the two Hb's were approximately equal, but after longer induction periods (3-7 da) Hb major was more abundant than Hb minor. In addition, it was found that the three proteases tested induced predominantly Hb minor. The addition of suboptimal concentrations of low molecular weight inducers acted synergistically with a given protease to produce a high yield of Hb-containing cells. When these agents were added singly they induced relatively low Hb major/Hb minor ratios, but when a low molecular weight inducer was added together with a protease in a "synergistic" combination, elevated ratios were induced. The proportions of hemoglobin types induced in MEL cells may be related in part to the intensity of the induction response. In view of these data, classifications of inducers based solely on the ratios of Hb types produced must be guarded.

The modulatory effect of 47 steroids on the DMSO stimulation of hemoglobin production in murine erythroleukemia cells was determined. The steroids capable of optimal inhibition of the DMSO effect most severely were glucocorticoids. Three steroids-halcinonide, flumethasone pivalate and flucloronide-were found to be active inhibitors at concentrations even lower than those noted for dexamethasone. Several steroids were noted to be capable of blocking the dexamethasone inhibition. Of these anti-inhibitors, fluoxymesterone and 17α-hydroxyprogesterone were the most active. The inhibitory and anti-inhibitory steroids were found to be the same steroid agonists or antagonists noted in other systems which are known to bind to glucocorticoid receptors. Therefore, it is likely that the activity of these steroids in erythroleukemia cell differentiation is mediated by typical glucocorticoid receptors. It was also shown that steroids strongly inhibit the DMSO effect only when present during DMSO stimulation of globin mRNA levels. This was further evidence that steroids were not acting at the level of translation. Since the same glucocorticoids that inhibit erythroid maturation in the mouse cells studied here also induce mouse lymphocytolysis and granulocytic differentiation, it is suggested that glucocorticoids may play an overall regulatory role in vivo in the determination of the numbers of mature blood cells of different types functioning in the organism. This type of corticosteroid action may occur in addition to any steroidal effects at earlier stages in hematopoiesis.

Friend leukemia cells grown in the presence of dimethyl sulfoxide show enhanced erythroid differentiation and hemoglobin synthesis. The effects of dimethyl sulfoxide stimulation are inhibited by BrdU. To determine the effect of BrdU on the amount of globin mRNA present in cells treated and not treated with dimethyl sulfoxide, molecular hybridization between total cell RNA and [ 3 H]DNA complementary to mouse globin mRNA was used. Cells treated with BrdU and dimethyl sulfoxide had 70% less globin mRNA than cells treated with dimethyl sulfoxide alone. The size and base sequence of the residual globin mRNA in the cultures treated with BrdU and dimethyl sulfoxide were unaltered. Cells treated with BrdU alone contained slightly more globin mRNA than did the untreated controls, suggesting that BrdU may have a dual effect in transcription of messenger RNA.

Differentiation along erythroid lines is greatly increased when Friend leukaemia cells (FLC) are grown in the presence of dimethylsulfoxide (DMSO). Maturation is accompanied by alterations in the polyribosome profiles of the DMSO-treated cells as compared to that of control cells grown for the same periods of time. The ribosomes of the differentiating cells are present primarily as trimers, tetramers and pentamers, with a low proportion of larger polysomes. Polyribosomes consisting of more than five ribosomes are much more common in the control untreated FLC. Agarose-acrylamide gel electrophoresis of radioactive uridine-labelled polysome-associated RNA from control and DMSO-treated cells demonstrates 4, 5, 5.5, 7, 9, 18 and 28S RNA species, as well as a series of RNAs which range from approximately 10S to 18S. The 5, 5.5, 18 and 28S RNAs have been tentatively identified as ribosomal RNAs, and the 4S and 7S RNAs may be transfer RNA and viral RNA respectively. The 9S RNA is probably the mRNA for histone, since its synthesis is inhibited in cells incubated with hydroxyurea. RNA which co-electrophoreses with the presumptive globin mRNA has been identified both in control, and in DMSO-treated cells. Although alterations in polyribosome patterns of DMSO-treated cells, as compared to control cells, have been readily demonstrable, no differences in the polyribosome-associated RNAs have been detected so far.

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.

Hemoglobin synthesis in murine Friend virus-induced leukemic cells established in tissue culture has been demonstrated. These cells have exhibited a limited degree of differentiation along the erythroid line throughout their serial passage history. Cloned lines differ in the extent of differentiation as evaluated by benzidine staining and the amount of hemoglobin produced. The hemoglobin present in these cells was identified by its histochemical staining properties, peroxidatic activity, incorporation of 59Fe, absorption spectrum, and characteristic migration pattern during discontinuous electrophoresis on polyacrylamide gels. Three hemoglobin components were detected on 10.0 per cent polyacrylamide gels. The electrophoretic pattern of these three hemoglobins appeared similar to that of hemolysates of adult DBA/2J mice, the strain from which the cells originated.

Tryptic peptides characteristic of mouse hemoglobin α- and diffuse-type β-chains were isolated from dimethylsulfoxide-treated mouse leukemia virus-infected cell cultures. Quantitative estimates of in vitro hemoglobin synthesis by such cultures were obtained by two different methods: by analysis of radioactive amino acid incorporation, and by single-cell radial immunodiffusion assay. The amounts produced, approximately 4 pg/cell/17 hr, reflect a synthetic capacity that is probably sufficient for 32P labeling of hemoglobin messenger RNAs (mRNA) in quantities commensurate with structural characterization of mRNAs by radioautographic methods.

Cells of a cloned line of murine virus-induced erythroleukemia were stimulated to differentiate along the erythroid pathway by dimethyl sulfoxide at concentrations that did not inhibit growth. A rise in the number of benzidine-positive normoblasts was accompanied by increased synthesis of heme and hemoglobin and a decrease in the malignancy of the cells. This action of dimethyl sulfoxide, which was reversible, may represent the derepression of leukemic cells to permit their maturation.

Seventeen different commercially available proteases stimulate both mouse erythroleukemia (MEL) cell differentiation and multiplication. These are the first enzymes shown to stimulate these processes in Friend leukemia virus-infected MEL cells. The induction of differentiation by proteases can be synergistically enhanced by the addition of low concentrations of dimethyl sylfoxide or other low molecular weight inducers. Since proteases are the first inducers of differentiation with a known biochemical function, their study should facilitate the understanding of the molecular mechanism of this process.

Erythrodifferentiation and hemoglobin synthesis in dimethyl sulfoxide-stimulated Friend erythroleukemia cells were inhibited by hydrocortisone (HC) and four other steroids: dexamethasone, deoxycorticosterone, corticosterone, and aldosterone. The effect was specific, because no significant cytotoxicity occurred with any of these compounds at the concentrations that were inhibitory. The mechanism of action of HC was studied in detail. In the absence of dimethyl sulfoxide, it had no effect on hemoglobin levels; but, in the presence of this inducer, the synthesis of heme and globin were each inhibited by approximately 90%. There was no alteration in the synthesis of any major protein other than globin, as determined by gel electrophoresis of cell lysates. The activities of two enzymes in the heme biosynthetic pathway, delta-aminolevulinate dehydratase and uroporphyrinogen-I synthase, were inhibited by 80% and 70%, respectively. Globin mRNA induction was reduced by approximately 90%. This demonstrated that the HC inhibition of globin synthesis occurred at a pretranslational step. The dimethyl sulfoxide-induced single-stranded breaks in DNA, which have been suggested to play a role in Friend leukemia cell differentiation, were reduced in number but not eliminated. HC reduced the dimethyl sulfoxide-stimulation of virus release into the medium by approximately 50%. HC treatment in the absence of dimethyl sulfoxide doubled the production of virus.

The tissues of the uterus, like most tissues, are composed chiefly of water; only one-fifth of the total weight is contributed by other substances. Proteins form the bulk of the solid material, constituting approximately 80% of the formed matter. The next most prominent uterine constituent by weight is deoxyribonucleic acid (DNA), which accounts for about 8% of the dry weight. DNA is followed in order by phospholipid, ribonucleic acid (RNA), and neutral fat. The remaining uterine components total approximately 5% of the dry weight and less than 1% of the total weight. A general outline of the chemical composition of the rat uterus is given in Table 1. It may be noted that many compounds, including intermediates of the glycolytic and citric acid cycles, some electrolytes, free amino acids, some mucopolysaccharides, and many other trace organic compounds have not been included in Table 1. A summary of the chemical composition of the uterus, based on several species in different stages of estrus and the menstrual cycle, has been compiled by Kosterlitz (1961).

Proteases play critical roles in a vast number of reactions that are important in regulating anabolic as well as catabolic cellular metabolism and, in addition, many extra- and intracellular physiologic functions (158, 165, 288). Although regulation of specific and general protein levels by catabolism was not as emphasized in early studies as that of the regulation of protein synthesis, this is now recognized as an error (4, 200). The importance of proteases in “molding” a final protein product by converting a zymogen, a pro-hormone, or a pro-growth factor to an active moiety, or by removing a signal peptide to have the protein “delivered” to the proper location, is clear (see Refs. 295, 338, 397). Specific examples include the activation of epidermal growth factor (EGF) by the proteolytic EGF-binding protein (123, 384) or the auto-proteolytic processing of pro-β nerve growth factor (NGF) to NGF by its own γ-subunit (142, 389). The importance of studying protease induction of cell proliferation and DNA synthesis is now recognized (see Refs. 265, 266 for earlier reviews) and it has also been suggested that proteases may act as growth factors for malignant cells (41, 254) or in wound healing (see Ref. 30). Proteases appear to be able to act as true growth factors and not simply in a process directed toward supplying nutrient amino acids, as carefully defined by Gospodarowicz and Moran (140), although the latter case has not been ruled out as an augmenting process.

Lines DS-19 and 5-86, each derived from line 745, when tested for their responses to various inducers and inhibitors of differentiation, shared some characteristics, but differed in others. In particular, DS-19 was markedly induced to differentiate by actinomycin D whereas 5-86 was only slightly affected. The patterns of the ability of PMA to influence induction and cell multiplication by various inducing agents differed in the two lines. The pattern of DEX inhibition of differentiation was similar in the two lines. Notably, DEX markedly inhibited induction due to all of the inducers tested except protease V8, actinomycin D, PGE1, and butyrate and its fatty acid analogues that were tested. DEX stimulated growth during its inhibition of induction by DMSO and many other inducers, but reduced cell multiplication in the presence of butyrate. PMA inhibited induction by most of the inducers tested in DS-19 cells except for some of the fatty acids. The inhibition by PMA generally was accompanied by cytotoxicity in DS-19 cells, but not in 5-86 cells. PMA markedly inhibited differentiation by only 5 of the inducing agents tested in 5-86 cells, but was not as cytotoxic in this line. Proteases, which have been shown to stimulate both MEL cell differentiation and growth, are inhibited with respect to their effects on differentiation, but not with respect to those on growth by DEX and/or PMA. DEX did not have the same effect on induction stimulated by the two proteases studied. Many of these findings indicate that at least some effects by inducers on cell multiplication in this system are not inextricably linked to differentiation. It is hoped that the further study of induction by proteases, which have known enzymatic activities, as well as of inhibitors of induction, will shed light on the molecular mechanism(s) of action of DMSO and other low molecular weight inducers.

The rate of DNA synthesis in secondary mouse embryo fibroblast (MEF-2) cultures was increased following infection with Friend leukemai virus (FLV) as compared to control cultures which were untreated or exposed to heat-denatured virus. A peak was reached between 24 and 48 h after infection. Radioautographic studies revealed that the infected cultures had a higher percentage of cells which incorporated tritiated thymidine into their nuclei as compared to controls. This increase was apparently independent of the ability of the cells to divide, since it was also observed in infected stationary cultures. The rates of cellular RNA and protein synthesis did not appear to be affected by FLV infection. The time required for the cell population to double was one-third shorter in FLV-infected cultures as compared to controls. The shortening of the generation time was found to be due to a decrease in the time required for the cells to traverse the G1 and S phases of the cell cycle. In addition, after 72 h of growth, the infected cultures reached greater population densities than did either of the controls. Alterations de la Synthese Macromoleculaire et de la Croissance Cellulaire dans les Fibroblastes D'Embryon de Souris Infectes par le Virus de la Leucemie de Friend Les auteurs ont constate que, par comparaison avec les cultures temoins non traitees ou exposees a un virus denature par la chaleur, le taux de synthese de l'ADN dans les cultures secondaires de fibroblastes d'embryon de souris (MEF-2) s'est accru apres infection par le virus de la leucemie de Friend (FLV). Un pic a ete atteint 24 a 48 h apres l'infection. L'autoradiographie a revele que les cultures infectees contenaient un pourcentage plus eleve de cellules incorporant la thymidine tritiee a leur noyau que les cultures temoins. Cet accroissement ne semblait pas dependre de l'aptitude des cellules a se diviser, puisqu'on l'a aussi observe dans les cultures stationnaires infectees. Le taux de synthese des proteines et de l'ARN cellulaires ne semble pas affecte par l'infection due au FLV. Le temps necessaire a la population cellulaire pour doubler etait plus court d'un tiers dans les cultures infectees par le FLV que chez les temoins. On a constate que le raccourcissement du temps de generation est dǔ au fait que les cellules mettent moins longtemps a traverser les phases G1 et S du cycle cellulaire. En outre, apres 72 heures de croissance, les densites de population etaient plus grandes dans les cultures infectees que chez les temoins.

Publisher Summary The chapter discusses about the murine virus-induced leukemia (FLV) that serves as a model system for exploring the relationship between differentiation and malignancy. The cells of this virus-induced leukemia (FLC) differentiate along the erythroid pathway in vitro and, under special conditions, in vivo as well. Reports on the differentiation of tumor cells in vitro include: studies of human and murine neuroblastoma cells as well as of human leukemic cells and murine leukemic cells. The studies describe that the malignant cells possess at least some of the genetic information that controls the growth and fate of their normal counterparts. Recent studies on human acute lymphoblastic leukemia cells demonstrate the presence of terminal deoxynucleotidyl transferase, an enzyme found only in normal thymus cells, indicating that in some human leukemic cells, there is retention of certain specific genetic information of the tissue of origin. It was observed that FLC, cultured in the presence of DMSO or DMF alone or in combination with BUdR, increased numbers of budding viruses on their cell membranes. It appeared that the cells synthesizing murine leukemia Type C virus particles are not necessarily malignant, for the agents (DMSO, DMF) that stimulate differentiation, with a concomitant decrease in malignant potential of FLC, also increased the number of budding virus particles. BUdR inhibits the differentiation and, yet, also increases the number of budding viruses.