Removal of ciprofloxacin from seawater by reverse osmosis.

Much of the deterioration of water resources is anthropogenically caused as a consequence of the incessant production of chemical compounds to obtain the quality of life that society demands today. This constant presence and harmful accumulation of these pollutants in different ecosystems have seen them emerge as a major concern both for human health and for environmental safety. Scientific advances have succeeded in legislating against, reducing and even eliminating priority pollutants, while new technologies are being constantly developed to identify and treat newly emerging pollutants. The objective of this work is the evaluation of the seawater reverse osmosis membrane as a method for the removal of an antibiotic present in seawater. The novelty of the study is that the tests were undertaken using water of high ionic strength. A critical selection of the antibiotic to be used in the study was carried out. The experiments were performed under constant pressure conditions, employing synthetic seawater in a pilot-scale unit with a commercial spiral-wound reverse osmosis membrane. Results are shown in terms of selectivity of the reverse osmosis process for antibiotic removal. The RO membrane element successfully reject most of the ciprofloxacin (removal rate >90%), with maximum rejection value of 99.96%.

The Ski proto-oncogene regulates body composition and suppresses lipogenesis.

OBJECTIVE: The Ski gene regulates skeletal muscle differentiation in vitro and and in vivo. In the c-Ski overexpression mouse model there occurs marked skeletal muscle hypertrophy with decreased adipose tissue mass. In this study, we have investigated the underlying molecular mechanisms responsible for the increased skeletal muscle and decreased adipose tissue mass in the c-Ski mouse. APPROACH: Growth and body composition analysis (tissue weights and dual energy X-ray absorptiometry) coupled with skeletal muscle and white adipose gene expression and metabolic phenotyping in c-Ski mice and wild-type (WT) littermate controls was performed. RESULTS: The growth and body composition studies confirmed the early onset of accelerated body growth, with increased lean mass and decreased fat mass in the c-Ski mice. Gene expression analysis in skeletal muscle from c-Ski mice compared with WT mice showed significant differences in myogenic and lipogenic gene expressions that are consistent with the body composition phenotype. Skeletal muscle of c-Ski mice had significantly repressed Smad1, 4, 7 and myostatin gene expression and elevated myogenin, myocyte enhancer factor 2, insulin-like growth factor-1 receptor and insulin-like growth factor-2 expression. Strikingly, expression of the mRNAs encoding the master lipogenic regulators, sterol-regulatory enhancer binding protein 1c (SREBP1c), and the nuclear receptor liver X-receptor-alpha, and their downstream target genes, SCD-1 and FAS, were suppressed in skeletal muscle of c-Ski mice, as were the expressions of other nuclear receptors involved in adipogenesis and metabolism, such as peroxisome proliferator-activated receptor-gamma, glucocorticoid receptor and retinoic acid receptor-related orphan receptor-alpha. Transfection analysis demonstrated Ski repressed the SREBP1c promoter. Moreover, palmitate oxidation and oxidative enzyme activity was increased in skeletal muscle of c-Ski mice. These results suggest that the Ski phenotype involves attenuated lipogenesis, decreased myostatin signalling, coupled to increased myogenesis and fatty acid oxidation. CONCLUSION: Ski regulates several genetic programs and signalling pathways that regulate skeletal muscle and adipose mass to influence body composition development, suggesting that Ski may have a role in risk for obesity and metabolic disease.

Prediction of radiosensitivity by measurement of X-ray induced apoptosis in human blood using the comet assay.

BACKGROUND: The aim of this work was to determine whether levels of radiation-induced apoptosis in human peripheral leukocytes could be used as a predictor of radiosensitivity. MATERIALS AND METHODS: Peripheral blood was obtained from venous blood and exposed to 0-3 Gy of X-rays. Apoptosis levels were measured at 4, 24, 48 and 72 hours after exposure using the neutral comet assay. Intra-individual apoptotic response was measured using repeated blood samples from four healthy individuals. Inter-individual variation was investigated in whole blood, granulocytes and mononuclear cells from 8 radiotherapy patients (4 demonstrating a radiosensitive response and 4 demonstrating a normal response to radiation exposures). RESULTS: Amongst the four healthy individuals there was both inter- and intra-individual variation of about the same magnitude. However, when comparing the apoptotic response of the radiosensitive and normal patients, consistent trends were observed at all X-ray doses for all of the patients. CONCLUSION: This indicates that apoptosis has some potential as a predictive assay, however, large intra-individual variation exists. More studies are required to investigate the causes of intra-individual variation and how it might be minimized.

Rubella susceptibility predicts measles susceptibility: implications for postpartum immunization.

Measles and mumps antibody titers were measured in 262 pregnant women who were either positive (n=128) or negative (n=134) for rubella antibodies. Susceptibility to measles and mumps was detected in 4.6% (12/262) and 7.6% (14/184) of the women, respectively. Of the rubella-susceptible group, 8.2% were also measles susceptible, whereas only 0.8% of the rubella-immune women were measles susceptible. Susceptibility to mumps was evenly divided between rubella-susceptible (7.8%) and rubella-immune (7.4%) groups.

Development and durability of measles antigen-specific lymphoproliferative response after MMR vaccination.

The correlates of long-term protection from measles infection are poorly understood. We followed the development of measles-specific antibody and lymphoproliferative (LP) responses in 60 children for 6 months after MMR vaccination. Prevaccine plaque reduction neutralization antibody (PRN Ab) values were low (mean+/-SEM 9.9+/-1. 1). Ninety-three percent (56/60) had excellent PRN values at 6 months (PRN 1816+/-207). Prevaccine LP activity was also low (stimulation index (SI)=1.4+/-0.1) but increased rapidly (SI 10. 7+/-4.5 at 2-3 weeks; p<0.05). However, only 61% (37/60) of the children had both significant cellular and antibody responses (SI>/=3 and PRN>/=120: Ab(hi)CMI(hi)). One child had a strong LP response (SI=6.7) despite little antibody production (PRN=19 at 6 months: Ab(lo)CMI(hi)). We also conducted a cross-sectional study in a separate group of 87 children 5-13 years after MMR vaccination. PRN values >/=120 were present in most children at 5-8 (n=28) and 9-13 years (n=59) after vaccination (PRN 550+/-120 and 360+/-60, respectively) but a significant minority had either undetected or 'subprotective' values (29 and 34%, respectively). LP responses (SI>/=3) were detectable in 19/28 (66%) and 36/59 (56%) of the children 5-8 and 9-13 years after vaccination (SI 11.4+/-2.4 and 7. 75+/-1.9, respectively). Almost two thirds (18/28) of the children in the cross-sectional study with low or absent antibody titers (PRN 41+/-6) had strong LP responses to measles antigens (SI 6.8+/-1.3). These data suggest that LP responses may be better sustained than antibody titers in some children. The susceptibility of Ab(lo)CMI(hi) children to infection and the value of the early LP response for predicting the durability of immunity remain to be determined.

Measurement of measles virus-specific neutralizing antibodies: evaluation of the syncytium inhibition assay in comparison with the plaque reduction neutralization test.

Plaque reduction neutralization (PRN) is the "gold-standard" for the measurement of measles-specific neutralizing antibodies. However, it is a complicated assay and tends to be operator-dependent. It has been suggested that the simpler syncytium inhibition assay (SIA) can give results comparable to the PRN test. We compared these two assays using 594 serum or plasma samples obtained from children at various times after natural infection, primary measles immunization, and measles revaccination. The results of the two assays correlated well overall (r = .86; p < 0.0001). The strain of challenge virus (wild-type versus vaccine strain) did not significantly influence SIA titers and the assay performed equally well with serum and plasma. PRN titers > or = 120 and > 800 are thought to indicate protection against clinical illness and infection respectively. The equivalent SIA cut-off values using 125 plaque-forming units as the challenge inoculum were > or = 16 and > 128 respectively. At low PRN titers (< 200), the correlation between PRN and SIA values was reasonable (r = 0.60; p < 0.001) when a challenge inoculum of 12.5 plaque-forming units was used. At the lowest PRN titers (< 100), 15% of the samples gave divergent results. These data confirm the utility of the SIA in the determination of measles-specific neutralizing antibodies when antibody titers are high. However, the PRN assay remains the test of choice when maximum sensitivity at low titers is required.

Growth arrest of immortalized human keratinocytes and suppression of telomerase activity by p21WAF1 gene expression.

Because most non-melanocytic human skin cancers have p53 mutations, it is unclear whether the aberrant growth of these cancers is simply a result of the abrogation of a p53 downstream mediator, the universal cyclin-dependent kinase inhibitor p21WAF1. To investigate the role of p21WAF1 in human skin carcinogenesis, we studied its regulation in normal and p53-mutated immortalized human keratinocytes. In proliferating human normal keratinocytes (HNK), more wild-type p53 protein (wt p53) was expressed than in growth-arrested differentiating keratinocytes. However, the function of wt p53 as a transcriptional activator of the p21WAF1 gene was suppressed in proliferating keratinocytes. In response to ultraviolet B irradiation, expression of wt p53 increased in proliferating keratinocytes, but p21WAF1 transcriptional activation was not induced. Two isoforms of mdm2 (p57 and p90), which can bind to wt p53 and negatively regulate wt p53 function, were expressed in proliferating HNK, suggesting that mdm2 may play a role in the suppression of wt p53's function in proliferating HNK. Increased expression of p21WAF1 was detected in both Ca(2+)-induced growth-arrested and differentiating HNK, in which the wt p53 expression was down regulated. This reflects the complexity of the p53/p21WAF1 pathways of cell-cycle regulation and differentiation in keratinocytes. No p21WAF1 expression was detected in human immortalized keratinocytes (HaCaT) or in two ras-transformed variants, HaCaT ras I/7 and HaCaT ras II/3, which have two p53 mutations. Retrovirus-mediated expression of p21WAF1 stopped the growth of all these cell types, but expression of wt p53 did not affect the cells' growth properties. p21WAF1 also downregulated human telomerase RNA component mRNA expression in HaCaT cells. This novel function of p21WAF1 partly explains the suppression of telomerase activity by p21WAF1 expression in HaCaT. Taken together, these results are consistent with the idea that p21WAF1 successfully inhibits the growth of non-melanocytic skin cancers, even those with alterations in p53, p21ras, retinoblastoma gene product, and telomerase activity.

Proliferation-associated regulation of telomerase activity in human endometrium and its potential implication in early cancer diagnosis.

Telomerase activity was detected in normal endometrium in association with proliferation and regulated during the menstrual cycle in a hormone-dependent manner. The activity was maximal at the late-proliferative phase to mid-secreting phase, and was absent or extremely low at early-proliferative phase and late-secreting phase. Activity was also detected in all endometrial simple hyperplasias tested (16 of 16) and in most cancers (28 of 30), but none was detected in endometrium of either pregnant or postmenopausal women in the absence of hyperplasia. Our data provide evidence that the telomerase activity in postmenopausal endometrium reflects a hyperproliferative condition. Therefore, we conclude that telomerase can provide a novel marker for early endometrial cancer diagnosis. Hormone-dependent regulation of telomerase suggests the possibility of therapeutic and preventive strategies for endometrial cancers through the management of ovarian steroid hormones or other agents that regulate telomerase activity.

Acquisition of heme iron by Neisseria meningitidis does not involve meningococcal transferrin-binding proteins.

Similarities in size between hemin-binding protein 1 (HmBP1) and transferrin-binding protein 1 (TBP1) of Neisseria meningitidis suggest that these proteins are functionally homologous. However, a meningococcal mutant lacking the transferrin-binding proteins retained the capacity to acquire iron from heme and hemoglobin. In immunoblots, hyperimmune polyclonal antiserum against TBP1 did not react with HmBP1.

UV and skin cancer: specific p53 gene mutation in normal skin as a biologically relevant exposure measurement.

Many human skin tumors contain mutated p53 genes that probably result from UV exposure. To investigate the link between UV exposure and p53 gene mutation, we developed two methods to detect presumptive UV-specific p53 gene mutations in UV-exposed normal skin. The methods are based on mutant allele-specific PCRs and ligase chain reactions and designed to detect CC to TT mutations at codons 245 and 247/248, using 10 micrograms of DNA samples. These specific mutations in the p53 gene have been reported in skin tumors. CC to TT mutations in the p53 gene were detected in cultured human skin cells only after UV irradiation, and the mutation frequency increased with increasing UV dose. Seventeen of 23 samples of normal skin from sun-exposed sites (74%) on Australian skin cancer patients contained CC to TT mutations in one or both of codons 245 and 247/248 of the p53 gene, and only 1 of 20 samples from non-sun-exposed sites (5%) harbored the mutation. None of 15 biopsies of normal skin from non-sun-exposed or intermittently exposed sites on volunteers living in France carried such mutations. Our results suggest that specific p53 gene mutations associated with human skin cancer are induced in normal skin by solar UV radiation. Measurement of these mutations may be useful as a biologically relevant measure of UV exposure in humans and as a possible predictor of risk for skin cancer.

Possible role of activated ras genes in human esophageal carcinogenesis.

Activation by point mutation of the H-, K- and N-ras genes is found in many tumors. However, no such mutation has yet been found in human esophageal carcinomas from various parts of the world. We have confirmed the absence of mutation at codons 12, 13 and 61 of K- and N-ras and at codons 12 and 61 of H-ras in 25 primary tumors obtained in France. In contrast, among 7 human esophageal carcinoma cell lines (TE1, TE2, TE3, TE8, TE9, TE10, TE13) with different degrees of tumorigenicity in nude mice, 3 highly tumorigenic lines (TE1, TE2 and TE8) exhibited activation of ras oncogenes; 2 showed a G35 to A35 transition of K-ras gene and one a H-ras G35 to T35 transversion. Since these cell lines had been established from tumors of Japanese patients from Sendai, we examined 3 primary esophageal tumors from Tokyo and 19 from Sendai, including the primary tumors from which the TE cell lines had been derived. No ras mutation was detected, which suggests that the ras gene mutations in the TE cell lines are either due to their long-term culture or that only a small portion of the original tumors contained such mutations. In order to directly examine the effect of ras gene mutation, one of the non-tumorigenic cell lines, TE13, was transfected with a plasmid coding for a mutated H-ras gene (G35 to T35). Transfected clones expressing high levels of mutated ras gene were able to induce tumors in nude mice. Thus, although no primary human esophageal tumor contained mutated ras genes, our studies do not exclude a significant role of mutated ras genes in cell proliferation and malignant transformation of human esophageal cells.

Transplacental and transgeneration carcinogenic effect of 7,12-dimethylbenz[a]anthracene: relationship with ras oncogene activation.

Transgeneration transmission of the carcinogenic action of 7,12-dimethylbenz[a]anthracene (DMBA) was studied in two generations of mice using transplacental DMBA initiation followed by postnatal skin tumor promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) in the first generation (F0) and only promotion in the second generation (F1). Local application of TPA resulted in increased skin tumor yield in both the in utero DMBA-exposed mice and their progeny (P = 0.0002 and P = 0.0941 respectively compared to control). Similarly, lung tumor incidence was increased in the two generations of mice (P less than 0.0001 and P = 0.0080 respectively). The results suggest transgeneration transfer of the effect of DMBA. A to T mutation at the second base of codon 61 of the Ha-ras oncogene was found in skin tumors of DMBA-exposed mice, but not in tumors induced by TPA without initiation. Analysis of Ki-ras codon 61 in seven lung tumors from DMBA-treated mice revealed three types of mutation: two cases with CA[C or G or T], one case with CCA and one case with CTA (the remaining cases having only the wild type). Six of these mice also had skin tumors, which contained A to T mutation at the second base of codon 61 of the Ha-ras gene in five cases. Thus mutations of different ras genes were found in skin and lung tumors from the same animals. In the progeny (F1) of DMBA-exposed F0 mice, only skin tumor samples were available for oncogene analysis and none contained the Ha-ras mutation. The results confirm our previous finding that initiation of skin and lung tumorigenesis can be transmitted transgenerationally. On the other hand, our data from a limited number of skin tumors suggests that ras gene mutation may not be critically involved in this transmission.

[Activation of the Ha-ras oncogene in tumors induced in mice by transplacental exposure to 7,12-dimethylbenz(a)anthracene]. / Aktivatsiia onkogena Ha-ras v opukholiakh, indutsirovannykh u myshei transplatsentarnym vozdeistviem 7,12-dimetilbenz(a)antratsena.

A study of tumors induced in mice by transplacental exposure to 7,12-dimethylbenz(a)anthracene (DMBA) alone or in combination with postnatal tissue-specific promotion showed skin and liver tumor development to be associated with cellular Ha-ras oncogene activation in a large percentage of cases. As shown by Xba I RFLP, oncogene activation was caused by T for A substitution at the second position of codon 61. The said mutation was traced in DMBA-induced tumors of the liver alone but not in spontaneous hepatomas. The results of the study showed the role of oncogene activation in cancer development to be tissue-specific.

Tissue-specific activating mutations of Ha- and Ki-ras oncogenes in skin, lung, and liver tumors induced in mice following transplacental exposure to DMBA.

Transplacental carcinogenesis represents a good model in which to study the involvement of tissue-specific oncogene activation in carcinogenesis because a single exposure to a carcinogen induces tumors at various sites. We tested tumors of the skin, liver, and lung produced in mice after transplacental 7,12-dimethylbenz[a]-anthracene (DMBA) exposure for possible activation of ras genes. XbaI restriction fragment-length polymorphism analysis has shown that exposure to DMBA in utero may result in appearance of A----T transversion at the second position of codon 61 of Ha-ras oncogene in skin and liver tumors but not in lung tumors. Moreover, DNA samples isolated from spontaneous and DMBA-induced lung and liver tumors were analyzed for mutations at the same position of Ki-ras oncogene using differential hybridization with specific oligonucleotides. Among five spontaneous lung tumors, three cases of A----G transition, and one case of A----T transversion were found, whereas four of ten lung tumors of DMBA-treated animals were positive for A----T mutation. No Ki-ras mutation was detected in one spontaneous and four DMBA-induced hepatomas. In two cases, we revealed Ki-ras A----T mutation in the lung tumor and Ha-ras mutation in the liver tumor taken from the same animal. These results indicate first that DMBA treatment may induce A----T mutation at the second position of codon 61 both in Ha-ras and in Ki-ras and, second, that the role of different activated oncogenes in carcinogenesis may differ, depending on the tissue in which the tumor develops.

Transplacental induction of a specific mutation in fetal Ha-ras and its critical role in post-natal carcinogenesis.

Mouse skin tumors were produced after transplacental initiation [with 7,12-dimethylbenz(a)anthracene], only when the skin was treated post-natally with a tumor-promoting agent (12-O-tetradecanoyl phorbol 13-acetate). DNA analysis of tumors showed that all carcinomas analyzed contained a specific mutation (A to T transversion) at the 61st codon of c-Ha-ras. Fifty per cent of the papillomas analyzed also had this same mutation. The A to T transversion at the 61st codon of Ha-ras was heterozygous in all positive papillomas and carcinomas. No such mutation was found when benzo(a)pyrene was used as an initiating agent. These results indicate that fetal c-Ha-ras can be transplacentally activated through a specific point mutation by a carcinogen, but a cell harboring such a mutation may remain dormant until it encounters a tumor-promoting stimulus.

Selective lack of intercellular communication between transformed and nontransformed cells as a common property of chemical and oncogene transformation of BALB/c 3T3 cells.

BALB/c 3T3 cells can be transformed by transfection of an activated cellular oncogene as well as by chemicals. When the cells were transformed by pEJ-ras transfection, a marked increase in Mr 21,000 protein expression was found by Western blotting and immunohistochemical staining, whereas no such increase was detected in cells transformed by methylcholanthrene, suggesting two different molecular mechanisms. By directly microinjecting a fluorescent dye (Lucifer Yellow CH) into individual cells, we measured junctional intercellular communication among and between transformed and surrounding nontransformed cells. In both chemical and oncogene transformation studies, transformed cells and surrounding normal cells have similar capacities for gap-junctional communication, but there was complete lack of communication between transformed and nontransformed cells. When BALB/c 3T3 cells were transformed by methylcholanthrene initiation followed by phorbol ester promotion, again we saw no intercellular communication between transformed and nontransformed cells, suggesting that the observed selective communication block between transformed and nontransformed cells may be a general phenomenon in BALB/c 3T3 cells. These results indicate that selective lack of intercellular communication between transformed and surrounding normal cells may be an important phenomenon that separates transformed cells and nontransformed cells, permitting transformed cells to maintain autonomous growth.

Comparative effects of a complete tumor promoter, TPA, and a second-stage tumor promoter, RPA, on intercellular communication, cell differentiation and cell transformation.

The biological activities in vitro of the incomplete (second-stage) tumor promoter, 12-O-retinoyl phorbol-13-acetate (RPA), and the complete tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) were compared. The doses of TPA and RPA necessary to inhibit the specific binding of [3H]-phorbol-12,13-dibutyrate ([3H]PDBu) to BALB/c 3T3 cells (50% inhibition doses; ID50; 8-13 ng/ml) were very similar; however, RPA was less potent than TPA in inhibiting [3H]-PDBu binding to Friend erythroleukemia cells (FELC). Intercellular communication between BALB/c 3T3 cells, measured by transfer of microinjected fluorescent dye (Lucifer Yellow), was inhibited by RPA as well as by TPA; TPA was about five times more potent than RPA. RPA also inhibited FELC differentiation induced by hexamethylene bisacetamide (HMBA) but not the differentiation of a TPA-resistant clone. The dose-responses of these two compounds in inhibiting differentiation of both TPA-sensitive and resistant FELC were very similar. When TPA and RPA were compared in their promoting activity of in vitro cell transformation of BALB/c 3T3 cells initiated with 3-methylcholanthrene (MCA, 0.1 microgram/ml), both TPA and RPA significantly increased the yield of morphologically transformed foci, and RPA was approximately 10 times more potent than TPA. These results suggest that RPA and TPA share many common in vitro biological effects and that these in vitro studies do not allow us to delineate clearly the effect of a second-stage tumor promoter from that of complete tumor promoters such as TPA.

Response of cultured rat liver epithelial cell lines to tumour-promoting phorbol esters.

The membrane effects of a potent tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), were studied in a series of cultured rat liver epithelial cell lines. Treatment with TPA resulted in the formation of strand-like aggregates (ridges) of viable cells over the monolayer of IAR 6-1 cells, but not of three other cell lines tested (IAR 20, IAR 6, IAR 6-7). The morphological response of IAR 6-1 to TPA was investigated by determination of phorbol ester receptors, analysis of cellular fucoproteins, surface galactoproteins and iodinatable surface proteins, and specific immunofluorescence for components of the extracellular matrix (fibronectin, laminin-entactin, procollagen type III). A class of specific, saturable, high-affinity receptors for phorbol esters was demonstrated in all four cell lines employing a conventional [20(-3)H]phorbol-12,13-dibutyrate ([3H]PDBu)-binding assay. The dissociation constants were similar in four lines, but the number of receptors per cell in IAR 6-1 cells was about twice that in other lines. Down-regulation of receptors was demonstrated in IAR 20 and IAR 6-1 cells with similar characteristics. Iodinatable surface proteins and galactose-containing surface glycoproteins did not respond to TPA. The distribution of fibronectin, laminin-entactin and procollagen type III was not affected by TPA. A TPA-responsive cell line, IAR 6-1, contained considerably less laminin-entactin than did the other lines. TPA had no influence on metabolic labelling of [3H]fucose-containing cellular glycoprotein in IAR 6-1 cells. One specific protein, with molecular mass of 78 kD, was more heavily labelled with [3H]fucose in IAR 6-1 cells than in the other cell lines. Taken together, the results of this study show that the responsive cells (IAR 6-1) differed from non-responsive ones in having more phorbol ester receptors, increased fucosylation of a specific glycoprotein and decreased deposition of laminin-entactin in the extracellular matrix. These surface properties of IAR 6-1 cells may contribute to their ability to respond to TPA.

Inhibition of cell communication between Balb/c 3T3 cells by tumor promoters and protection by cAMP.

The effect of phorbol ester tumor promoters on the communication between individual cells in confluent culture was studied using a fluorescent dye transfer method. Cell-cell communication between mouse Balb/c 3T3 cells and between Chinese hamster V79 cells was inhibited almost completely by tumor-promoting phorbol esters, but not by nonpromoting derivatives; the effect was reversed upon removal of the promoter. Intercellular communication between Balb/c 3T3 cells, but not Chinese hamster V79 cells, was increased significantly in the presence of dbcAMP and caffeine, and these compounds counteracted the effects of tumor promoters. Inhibition of cell communication by phorbol esters appears to be receptor-mediated, since specific binding of 3H-phorbol-12,13-dibutyrate to Balb/c 3T3 cells was inhibited only by compounds that also inhibit intercellular dye transfer. A study with cycloheximide suggests that the reversible inhibition of intercellular communication by phorbol esters may not need de novo protein synthesis, while upregulation of communication by cAMP requires protein synthesis.

Continuous suppression of globin gene expression and differentiation of Friend erythroleukemia cells by phorbol 12-myristate 13-acetate (PMA) despite the loss of PMA binding sites by down regulation.

The tumor promoter phorbol 12-myristate 13-acetate (PMA) reversibly inhibits hexamethylene bisacetamide-induced terminal differentiation of Friend erythroleukemia cells (FELC). We were successful in continuously inhibiting FELC differentiation by PMA up to 125 weeks (about 240 serial passages of cells in the presence of PMA). During that period, FELC can be induced to differentiate and enter terminal cell division upon removal of PMA. PMA-mediated suppression of FELC differentiation was associated with only a low level of globin mRNA accumulation. However, a rapid accumulation of globin mRNA in the cytoplasm followed by hemoglobin accumulation occurred upon removal of PMA. A specific, saturable, high-affinity receptor for phorbol esters is present in FELC, as was shown by binding studies with [3H]phorbol 12,13-dibutyrate. A significant (80%) loss in the number of phorbol ester receptors of FELC was observed after a continuous inhibition of differentiation by PMA for as much as 125 weeks. Despite such a down regulation of phorbol ester receptors, these cells respond to PMA with a dose-response similar to that of their parent cells, which have the normal number of phorbol ester receptors. Thus, PMA can suppress reversibly the accumulation of globin-specific mRNA and terminal differentiation of FELC during prolonged periods, despite loss of receptor sites, and our results suggest that only few phorbol ester receptors may be necessary for complete inhibition of FELC differentiation by PMA.