Retinoic acid enhances the displacement of newly synthesized hyaluronate from cell layer to culture medium during early phases of chondrogenesis

Chondrogenic differentiation in mouse limb bud mesenchymal cells cultured at high density was suppressed by supplementation of the medium with retinoic acid (1 microgram/ml or 3.3 X 10(-6) M). Since in control medium overt chondrogenesis begins on day 3, retinoic acid was introduced on day 2 so that the relationship between initial biosynthetic changes and inhibition of chondrogenesis could be studied. During the first 24 h of exposure the treated cells remained viable but suffered 10% inhibition in growth and synthesized [3H]glucosamine-labeled glycosaminoglycan at a level 24% below untreated cells. The amount of labeled hyaluronic acid released into the culture medium by the treated cells was, however, 2-fold greater, on a per cell basis, than that in the untreated cultures. It is suggested that the displacement of hyaluronate may play a role in the disruption of mesenchymal cell differentiation and of limb morphogenesis as observed in other systems.     

Effect of donor age and prior sun exposure on growth inhibition of cultured human dermal fibroblasts by all trans-retinoic acid

The effect of retinoic acid on human fibroblasts was studied in a cell culture model of chronologic aging and photoaging. During early exponential phase, all trans-retinoic acid significantly stimulated growth rate of adult arm-derived dermal fibroblasts but not of newborn or adult foreskin-derived fibroblasts. Retinoic acid also significantly reduced saturation density in most young adult arm-derived lines and all 24 lines derived from old adult arm and foreskin. However, four of ten young adult outer arm (relatively sun-exposed) and one of ten young adult inner arm (relatively sun-protected) fibroblasts lines increased their saturation density in response to retinoic acid. These data suggest that prior sun exposure and/or donor age may influence cellular responsiveness to retinoic acid. Neither the stimulatory nor the inhibitory effect of retinoic acid could be attributed to cell density, to breakdown of retinoic acid in culture, to nutrient depletion or to serum dependency. However, stationary phase fibroblasts from all sites (foreskin, inner and outer arm) showed an increase in filopodia and in intracellular actin after treatment with retinoic acid that was roughly proportional to the degree of growth inhibition, irrespective of donor age. We suggest that retinoic acid induces premature density dependent growth inhibition at least in part by increasing filopodia-mediated cell contact that is in turn directly related to an increase in fibrillar actin.     

Retinoic acid inhibition of transplasmalemma diferric transferrin reductase

All trans retinoic acid inhibited diferric transferrin reduction by HeLa cells. The NADH diferric transferrin reductase activity of isolated liver plasma membranes was also inhibited by retinoic acid. Retinol and retinyl acetate had very little effect. Transplasma membrane ferricyanide reduction by HeLa cells and NADH ferricyanide reductase of liver plasma membrane was also inhibited by retinoic acid, therefore the inhibition was in the electron transport system and not at the transferrin receptor. Since the transmembrane electron transport has been shown to stimulate cell growth, the growth inhibition by retinoic acid thus may be based on inhibition of the NADH diferric transferrin reductase.     

Retinoids and cultured human fibroblasts : Effects on cell growth and presence of cellular retinoic acid-binding protein

We have examined the effects of retinoids on growth of cultured human skin fibroblasts from four individuals. Retinoic acid and retinol both produce a dose-dependent inhibition of growth in the four strains examined; retinoic acid was more potent than retinol in this respect. The growth inhibitory effect of retinoic acid is characterized by a decrease in the exponential growth rate, which is reversible upon removal of retinoic acid from the growth medium; the final saturation density, however, is not modified by retinoic acid treatment. No alterations of cell morphology, viability, or adhesiveness to substratum are induced by the retinoid concentrations utilized. The inhibitory effect of 10⁻⁶ M retinoic acid on cell growth is not affected by the concentration of fetal calf serum (FCS) in the medium. In all four human fibroblast strains examined, specific binding of [³H]retinoic acid to cytosol is present as determined by sucrose-density gradient centrifugation. Despite the effects of retinol on fibroblast growth, no cytoplasmic binding of [³H]retinol could be demonstrated in these cells.     

Different susceptibilities of melanoma cells to retinoic acid-induced changes in melanotic expression

The effect of retinoic acid on murine B16 melanoma cell growth, tyrosinase activity and melanin synthesis was investigated. Retinoic acid inhibited the growth of B16F1, B16F10 and B16BL6 melanoma cells, but enhanced melanin synthesis only in the B16F1 cells. The B16F10 and B16BL6 cells exhibited retinoic acid-induced suppression of tyrosinase activity and melanin synthesis, which was most apparent in the B16F10 cell variant. For comparison, Cloudman S91 melanoma cells proved to be particularly sensitive to retinoic acid-induced growth inhibition and stimulation of the expression of their melanotic phenotype. These results suggest considerable heterogeneity in the B16 melanoma with respect to their response to retinoic acid.     

Effects of retinoic acid on cell proliferation and cell differentiation in a rat tracheal epithelial cell line

Abstract. The effects of 3.3 times 10-⁷ M to 3.3 times 10^-5 M all- trans -retinoic acid (vitamin-A acid) on the total cell population dynamics of 165 S, a keratinizing epithelial cell line from carcinogen-exposed rat trachea, were studied. During the first 6 days of culture, cells accumulated on the dish in the presence of the vitamin to twice the density of controls. [³H]thymidine incorporation into DNA and percentage of [³H]thymidine-labelled cells in autoradiographs were stimulated in a dose-dependent fashion to a maximum of 25- and 34-fold, respectively. Exfoliation of cells from the cultures was also enhanced 2–3-fold, resulting in nearly twice the total number of cells (attached plus exfoliated) in the presence of the vitamin.
During 19 days of culture, retinoic acid maintained a higher level of [³H]thymidine incorporation and cell exfoliation in 165 S cells so that by day 19, total cell production was more than three times that seen in controls. At this time, vitamin-treated cultures showed a reduced cell saturation density compared to controls. The higher final cell density in the control cultures was a result of multilayering and papillary formation which did not occur in the presence of retinoic acid. The papillae in control cultures stained specifically with Rhodamine B or with the eosin and orange G components of the Papanicolaou method. A count of the number of eosin and orange G positive cells in the attached and exfoliated cell compartments showed an 8-fold reduction of keratinization in retinoic acid-exposed cultures. Our results show that retinoic acid is a growth stimulant in these cell cultures, causing increased cell proliferation and exfoliation accompanied by inhibition of keratinization.     

Disabled-2 mediates c-Fos suppression and the cell growth regulatory activity of retinoic acid in embryonic carcinoma cells

F9 embryonic stem cell-like teratocarcinoma cells are widely used to study early embryonic development and cell differentiation. The cells can be induced by retinoic acid to undergo endodermal differentiation. The retinoic acid-induced differentiation accompanies cell growth suppression, and thus, F9 cells are also often used as a model for analysis of retinoic acid biological activity. We have recently shown that MAPK activation and c-Fos expression are uncoupled in F9 cells upon retinoic acid-induced endodermal differentiation. The expression of the candidate tumor suppressor Disabled-2 is induced and correlates with cell growth suppression in F9 cells. We were not able to establish stable Disabled-2 expression by cDNA transfection in F9 cells without induction of spontaneous cell differentiation. Transient transfection of Dab2 by adenoviral vector nevertheless suppresses Elk-1 phosphorylation, c-Fos expression, and cell growth. In PA-1, another teratocarcinoma cell line of human origin that has no or very low levels of Disabled-2, retinoic acid fails to induce Disabled-2, correlating with a lack of growth suppression, although PA-1 is responsive to retinoic acid in morphological change. Transfection and expression of Disabled-2 in PA-1 cells mimic the effects of retinoic acid on growth suppression; the Disabled-2-expressing cells reach a much lower saturation density, and serum-stimulated c-Fos expression is greatly suppressed and disassociated from MAPK activation. Thus, Dab2 is one of the principal genes induced by retinoic acid involved in cell growth suppression, and expression of Dab2 alone is sufficient for uncoupling of MAPK activation and c-Fos expression. Resistance to retinoic acid regulation in PA-1 cells likely results from defects in retinoic acid up-regulation of Dab2 expression.     

Induction of cell differentiation in melanoma cells by inhibitors of IMP dehydrogenase: altered patterns of IMP dehydrogenase expression and activity

To study the induction of differentiation in human melanoma cells, we treated 12 melanoma cell lines with mycophenolic acid and tiazofurin, inhibitors of IMP dehydrogenase (IMPDH). In all cell lines studied, both agents inhibited cell growth and increased melanin content. However, the degree of growth inhibition did not necessarily correspond to the increase in melanin content. A detailed analysis of the HO and SK-MEL-131 cell lines indicated that mycophenolic acid and tiazofurin caused a time- and dose-dependent increase in the expression of a series of other maturation markers, including formation of dendrite-like structures, tyrosinase activity, and reactivity with the CF21 monoclonal antibody. The growth inhibition and melanogenesis induced by the IMPDH inhibitors was abrogated by the addition of exogenous guanosine. No such effect was observed after treatment of the cells with phorbol 12-myristate 13-acetate or retinoic acid, two other inducers of differentiation in these cells. The mycophenolic acid- and tiazofurin-treated cells also showed an increased level of IMPDH mRNA and protein, perhaps because of compensation for the inhibitor-mediated decrease in IMPDH activity. In contrast, treatment with phorbol 12-myristate 13-acetate or retinoic acid resulted in decreased levels of IMPDH mRNA and protein. The lack of a consistent pattern of IMPDH expression in the cells treated with IMPDH inhibitors and phorbol 12-myristate 13-acetate or retinoic acid suggests that the altered expression of IMPDH is not a general requirement for the induction of cell differentiation in these cells. Our results also suggest that IMPDH inhibitors may provide a useful approach to circumvent the differentiation block in melanoma.     

Catalase-like oxygen production by horseradish peroxidase must predominantly be an enzyme-catalyzed reaction

The biochemical mechanisms underlying the inhibitory effects of lycopene, the main tomato carotenoid, on the growth of cancer cells are largely unknown. It has been hypothesized that lycopene derivatives may act as ligands for a nuclear receptor in analogy to retinoic acid, the hormone derived from beta-carotene. The inhibition of human mammary cancer (MCF-7) cell growth and the transactivation of the retinoic acid receptor (RAR) reporter gene by synthetic acyclo-retinoic acid, the open chain analog of retinoic acid, was compared to the effects of lycopene and retinoic acid in the same systems. Acyclo-retinoic acid activated the DR-5 retinoic acid response element with a approximately 100-fold lower potency than retinoic acid. This effect was independent of cotransfection with the RARalpha receptor. Lycopene exhibited only very modest activity in this system. In contrast to the results from the transactivation studies, acyclo-retinoic acid, retinoic acid, and lycopene inhibited cell growth with a similar potency. Preincubation with each of the three compounds slowed down cell cycle progression from G1 to S phase. In summary, acyclo-retinoic acid inhibited cancer cell growth and interacted with RAR. However, it exhibited low affinity for RAR and a correspondingly low efficacy in activating this receptor, indicating that RAR does not mediate the growth inhibitory effect of the compound. In addition, the concentrations of acyclo-retinoic acid and of lycopene required for inducing inhibition of cell growth were similar, suggesting that acyclo-retinoic acid is unlikely to be the active metabolite of lycopene.     

Retinoic acid-induced modifications in the growth and cell surface components of a human carcinoma (HeLa) cell line

The addition of retinoic acid to cultures of HeLa-S3 cells caused a reduction in cell proliferation rate which became apparent after 72 h and was linearly dependent on retinoic acid concentration in the range 10⁻⁹–10⁻⁵ M. After 72 h of exposure to retinoic acid, the cells assumed a flattened appearance and no longer formed multilayers. These changes were reversed within 48 h after removal of retinoic acid from the medium. Structural analogs of retinoic acid with a free ---COOH group at C-15 were usually more potent in growth inhibition than compounds with an alcohol, aldehyde, ether or ester group. A cellular retinoic acid-binding protein was detected in cell homogenates, and the binding of [³H]retinoic acid to the binding protein was inhibited by most, but not all, analogs possessing a free terminal ---COOH group. For example, the 4-oxo analog of retinoic acid, while capable of inhibiting cellular proliferation, failed to bind to the retinoic acid-binding protein. Analysis of cell surface and cellular glycoproteins by lactoperoxidase-catalysed ¹²⁵I iodination and by metabolic labeling with [³H]glucosamine revealed that a 190000 D glycoprotein which was labeled by both methods and a 230000 D glycoprotein which was labeled only with [³H]glucosamine were labeled more intensely in retinoic acid-treated cells compared with untreated cells. The electrophoretic mobility of the 230000 D glycoprotein could be modified by treatment of intact cells with either neuraminidase or proteolytic enzymes, suggesting that this glycoprotein is also exposed on the cell surface. The cell surface alterations were detected much earlier than the onset of growth inhibition and appeared as early as 24 h after exposure to retinoic acid. The possible relationship between retinoic acid-induced changes in cell membrane structure, cell morphology, and cell proliferation is discussed.     

Ectopic expression of CXCR5/BLR1 accelerates retinoic acid- and vitamin D(3)-induced monocytic differentiation of U937 cells

The product of the blr1 gene is a CXC chemokine receptor (CXCR5) that regulates B lymphocyte migration and has been implicated in myelomonocytic differentiation. The U937 human leukemia cell line was used to study the role of blr1 in retinoic acid-regulated monocytic leukemia cell growth and differentiation. blr1 mRNA expression was induced within 12 hr by retinoic acid in U937 cells. To determine whether the early induction of blr1 might regulate inducible monocytic cell differentiation, U937 cells were stably transfected with blr1 (U937/blr1 cells). Ectopic expression of blr1 caused no significant cell cycle or differentiation changes, but caused the U937/blr1 cells to differentiate faster when treated with either retinoic acid or 1alpha,25-dihydroxyvitamin D(3). Treated with retinoic acid, U937/blr1 cells showed a greater increase in the percentage of CD11b expressing cells than vector control cells. Retinoic acid also induced a higher percentage of functionally differentiated blr1 transfectants as assessed by nitroblue tetrazolium reduction. U937/blr1 cells underwent moderate growth inhibition on treatment with retinoic acid. Similar results occurred with 1alpha,25-dihydroxyvitamin D(3). Because blr1 was induced early during cell differentiation and because its overexpression accelerated monocytic differentiation, it may be important for signals controlling cell differentiation.     

Increased expression of cyclin A1 protein is associated with all-trans retinoic acid-induced apoptosis

Deregulated cell growth and inhibition of apoptosis are hallmarks of cancer. All-trans retinoic acid induces clinical remission in patients with acute promyelocytic leukemia by inhibiting cell growth and inducing differentiation and apoptosis of the leukemic blasts. An important role of the cell cycle regulatory protein, cyclin A1, in the development of acute myeloid leukemia has previously been demonstrated in a transgenic mouse model. We have recently shown that there was a direct interaction between cyclin A1 and a major all-trans retinoic acid receptor, RAR alpha, following all-trans retinoic acid treatment of leukemic cells. In the present study, we investigated whether cyclin A1 might be involved in all-trans retinoic acid-induced apoptosis in U-937 leukemic cells. We found that all-trans retinoic acid-induced apoptosis was associated with concomitant increase in cyclin A1 expression. However, there was no induction of cyclin A1 mRNA expression following the all-trans retinoic acid-induced apoptosis. Treatment of cells with a caspase inhibitor was not able to prevent all-trans retinoic acid-induced up-regulation of cyclin A1 expression. Interestingly, induced cyclin A1 expression in U-937 cells led to a significant increase in the proportion of apoptotic cells. Further, U-937 cells overexpressing cyclin A1 appeared to be more sensitive to all-trans retinoic acid-induced apoptosis indicating the ability of cyclin A1 to mediate all-trans retinoic acid-induced apoptosis. Induced cyclin E expression was not able to initiate cell death in U-937 cells. Our results indicate that cyclin A1 might have a role in apoptosis by mediating all-trans retinoic acid-induced apoptosis.     

The role of polypeptide growth factors in phenotypic transformation of normal rat kidney cells

A serum-free assay has been established for studying the role of polypeptide growth factors in inducing loss of density-dependent inhibition of growth of normal rat kidney (NRK) cells. The process has been characterized by measuring the time course of [3H]thymidine incorporation into confluent, quiescent NRK cultures stimulated by defined polypeptide growth factors, in combination with cell counting studies, increases in DNA content, and cell cycle analysis by means of a fluorescence-activated cell sorter. It is shown that none of the growth factors tested (epidermal growth factor, platelet-derived growth factor, transforming growth factor-beta, and retinoic acid) is able to induce loss of density-dependent inhibition of growth by itself, but strong synergism was observed when combinations of growth factors were tested. None of the above factors was found to be essential, however, since any combination of three of the above four growth factors strongly induced the process. Strong parallels were observed between the growth factor requirements for inducing loss of density-dependent inhibition of growth under serum-free conditions and the requirements for induction of anchorage-independent proliferation under growth factor-defined assay conditions. This indicates that most likely the same cellular processes underlie these two aspects of phenotypic transformation, although data indicate that anchorage-independent proliferation may be a more restricted property of phenotypic transformation than loss of density dependence of proliferation. It is concluded that phenotypic transformation of NRK cells does not require specific polypeptide growth factors, but reflects the ability of these cells to respond to multiple growth factors.     

Interaction of retinoids and tamoxifen on the inhibition of human mammary carcinoma cell proliferation

The growth of chemically induced mammary tumors is inhibited by both hormone manipulation as well as by retinoids. Numerous mammary carcinoma cell lines are also inhibited by retinoids. Co-treatment of estrogen receptor (ER)-positive breast cancer cells resulted in an additive effect in terms of inhibition of cellular proliferation. The addition of varying concentrations of retinoic acid (RA) to varying concentrations of tamoxifen (TMX) resulted in an additive effect on the inhibition of proliferation of the ER-positive human carcinoma cell lines (MCF-7). Co-treatment of MCF-7 cells over time with RA and TMX resulted in enhanced inhibition of growth. A similar phenomenon was observed when other synthetic retinoids were combined with TMX. This enhanced inhibition by the combination of retinoids and TMX was also observed with other ER-positive cell lines (ZR-75, T47-D), while no effect was noted on the ER-negative cell lines (MDA-MB-231, Hs578T).     

Participation of type II protein kinase A in the retinoic acid-induced growth inhibition of SH-SY5Y human neuroblastoma cells

To examine the role of protein kinase A (EC 2.7.1.37) isozymes in the retinoic acid-induced growth inhibition and neuronal differentiation, we investigated the changes of protein kinase A isozyme patterns in retinoic acid-treated SH-SY5Y human neuroblastoma cells. Retinoic acid induced growth inhibition and neuronal differentiation of SH-SY5Y cells in a dose- and time-dependent manner. Neuronal differentiation was evidenced by extensive neurite outgrowth, decrease of N-Myc oncoprotein, and increase of GAP-43 mRNA. Type II protein kinase A activity increased by 1.5-fold in differentiated SH-SY5Y cells by retinoic acid treatment. The increase of type II protein kinase A was due to the increase of RIIbeta and Calpha subunits. Since type II protein kinase A and RIIbeta have been known to play important role(s) in the growth inhibition and differentiation of cancer cells, we further investigated the role of the increased type II protein kinase A by overexpressing RIIbeta in SH-SY5Y cells. The growth of RIIbeta-overexpressing cells was slower than that of parental cells, being comparable to that of retinoic acid-treated cells. Retinoic acid treatment further increased the RIIbeta level and further inhibited the growth of RIIbeta-overexpressing cells, showing strong correlation between the level of RIIbeta and growth inhibition. However, RIIbeta-overexpressing cells did not show any sign of neuronal differentiation and responded to retinoic acid in the same way as parental cells. These data suggest that protein kinase A participates in the retinoic acid-induced growth inhibition through the up-regulation of RIIbeta/type II protein kinase A.     

Inhibition of epidermal growth factor receptor activity by retinoic acid in glioma cells

The growth inhibitory effects of exogenously added retinoic acid (RA) on various cultured human glioma cells was observed to be heterogenous, with an ID50 ranging from 10(-7) M to no response. The protein tyrosine kinase activity of epidermal growth factor receptor (EGF-receptor) appeared to parallel the cell's growth responsiveness to RA. Cells sensitive to RA-induced growth inhibition exhibited a dose-dependent decrease in EGF-receptor activity, whereas RA-resistant cells showed no alterations in EGF-receptor protein tyrosine kinase activity or expression. The modulation of EGF-receptor by RA was further examined with RA-sensitive (LG) and -resistant (NG-1) cell lines. Both cell lines were approximately equal in their ability to bind and internalize epidermal growth factor in the presence or absence of RA. Several independent assays suggested that the inhibition of EGF-receptor activity was independent of protein kinase C modulation as mediated by phorbol myristate acetate. However, alterations in associated glycoconjugates of EGF-receptor were observed among the sensitive cells but not the resistant cells. These results suggest RA-induced growth inhibition in sensitive cells may arise, at least in part, through alterations in EGF-receptor and structure.     

Differential effects of c-myb and c-fes antisense oligodeoxynucleotides on granulocytic differentiation of human myeloid leukemia HL60 cells

To gain some insight into the role of c-myb and c-fes in myeloid differentiation, the authors have analyzed the ability of HL60 cells to differentiate in response to several different inducers after inhibition of c-myb and c-fes function. This function has been inhibited almost completely by using deoxynucleotides complementary to two 18-nucleotide sequences of c-myb and c-fes encoding mRNA. After 5 days in culture, in several separate experiments with different oligomer preparations, more than 90% growth inhibition was observed in c-myb antisense-treated HL60 cells. At this time, independent of the differentiation inducer used, c-myb antisense-treated HL60 cells differentiate only along the monocytic pathway, whereas in sense oligomer-treated cultures, retinoic acid and dimethyl sulfoxide induced granulocytic differentiation. No perturbation of the HL60 cell growth was observed after 5 days of treatment with antisense c-fes oligomer. However, induction to granulocytic differentiation by retinoic acid and dimethyl sulfoxide resulted in progressive cell death, whereas monocytic differentiation by other differentiation inducers was only marginally affected. These results suggest that granulocytic, unlike monocytic, differentiation requires c-myb-conditioned proliferation and the activity of the protein encoded by c-fes.