Effect of laparoscopy versus laparotomy on circulating tumor cells using isolation by size of epithelial tumor cells.

AIM: To assess the effect of laparoscopy on circulating tumor cell (CTC) detection in case of carcinosis. MATERIAL AND METHODS: We compared laparoscopy versus laparotomy on tumor cell blood release in an animal model of ovarian carcinosis obtained by intraperitoneal inoculation of IGR-OV1 cells in nude rats. Animals were randomly assigned to one of the following groups: CO(2) laparoscopy (L), gasless laparoscopy (GL), midline laparotomy (ML), or general anesthesia as control (C). A 0.5 ml blood sample was taken in each case before and after experiment and tested with a novel assay, ISET (isolation by size of epithelial tumor cells), which isolates CTC by filtration on account of their size. Statistics were performed with the Fisher's and the Chi-square tests. RESULTS: Ten rats were included in each group. We did not find any significant difference in CTC prevalence before and after surgery (2/14 versus 3/19, respectively, P = 1). Similarly, the three surgical accesses were equivalent with one post-experiment detection per group: 1/5 for L, 1/7 for ML, 1/7 for GL, and 1/6 for C (P = 0.9). CONCLUSION: This trial did not show any deleterious effect of laparoscopy on CTC when compared to laparotomy.

Impact of pneumoperitoneum on tumor growth.

AIM OF THE STUDY: To compare intraperitoneal tumor growth after CO2 laparoscopy (L), gasless laparoscopy (GL), midline laparotomy (ML), and general anesthesia (GA) as a control. MATERIALS AND METHODS: A prospective randomized trial was carried out in nude rats. A carcinomatosis was obtained by intraperitoneal injection of either one of the two human ovarian cancer cell lines IGR-OV1 or NIH:OVCAR-3. Rats secondly underwent randomly different kind of procedures: CO2 L (8 mmHg, 60 min), GL (traction by a balloon for 60 min), ML (bowel removed and let on a mesh for 60 min), or GA. The rats were finally killed 10 or 35 days after surgery (respectively in IGR-OV1, or NIH:OVCAR-3 models). Tumor growth was assessed by the weight of the omental metastasis and MIB1 immunostaining. Peritoneal dissemination as well as abdominal wall metastases were assessed by pathological examination. Statistical analysis used the chi-square test (or Fisher exact test) and Bonferroni method for multiple comparison between groups. RESULTS: Fifteen rats were included in each group. Mean omental weight was significantly increased after surgery (3.1 to 5.6 g), when compared to control (2.4 g), but no significant difference was recorded between the three surgical accesses. MIB1 immunostaining was poor in the PNP group (37%), whereas it was higher after midline laparotomy (51%), but the difference was not significant (p = 0.07). Similarly, no significant variation was recorded in the NIH:OVCAR-3 model for omental weight or MIB1 staining. CO2 pneumoperitoneum significantly increased right diaphragmatic dome involvement in the NIH:OVCAR-3 model. Abdominal wall metastases were significantly more frequent after surgery when compared to the control group, but no significant difference could be demonstrated between surgical groups in each model. CONCLUSION: In these solid tumor models, CO2 pneumoperitoneum had no deleterious effect on tumor growth when compared to gasless laparoscopy or midline laparotomy.

Peritoneal closure reduces port site metastases: results of an experiment in a rat ovarian cancer model.

BACKGROUND: The arm of this study was to assess the role of peritoneal closure in the prevention of port site metastasis after carbon dioxide (CO2) CO2 pneumoperitoneum. METHODS: We developed a xenograft ovarian cancer model by intraperitoneal injection of 27 106 IGR-OV1 line cells in nude rats Seven days after the inoculation, the animals underwent a CO2 pneumoperitoneum. At the end of the procedure, port sites were randomly closed either with suture of peritoneum (n = 14, group A) or without suture of peritoneum (n = 12, group B). The rats were killed 7 days after surgery and their port site scars were resected. Tumor implantation was assessed by a pathologist who was blinded to the type of wound closure. RESULTS: The animals in group B were significantly more likely to have at least one port site metastasis frequent (seven of 12, or 58.3%) than those in group A (two of 14, or (14.3%) (p = 0.037). Port sites with metastases were seen more frequently in group B (eight of 24, or (33.3%) than in group A (three of 28, or 10.7%) (p = 0.046). CONCLUSIONS: Our results shows that peritoneum closure decreases the risk of port site metastasis.

Impact of different gases and pneumoperitoneum pressures on tumor growth during laparoscopy in a rat model.

BACKGROUND: The influence on intraperitoneal tumor growth of the choice of gas and pneumoperitoneum pressure during laparoscopy is still unknown. This study compared tumor growth after laparoscopy with different gases and pneumoperitoneum pressures in an immunodeficient model. METHODS: In an initial experiment, 60 nude rats were randomly allocated to undergo laparoscopy at different pneumoperitoneum pressures (gasless, 4 mmHg, or 8 mm Hg.) In a second experiment, 23 nude rats were randomly allocated to undergo laparoscopy with different gases (carbon dioxide or helium). Surgery was carried out 7 days after intraperitoneal injection of IGR-OV1 cells. The rats were killed 7 days after surgery. Tumor growth was assessed by the weight of the omental metastasis. For statistical analysis, we used analysis of variance (ANOVA). RESULTS: Mean omental weight was similar for all groups, regardless of the pneumoperitoneum pressure (p = 0.86) or the type of gas (p = 0.80). CONCLUSION: Physical parameters of gas have a limited impact on tumor growth.

Influence of peritoneal factors on port-site metastases in a xenograft ovarian cancer model.

OBJECTIVE: We determine the main physical effects of pneumoperitoneum on tumour dissemination and port-site metastases occurrence. DESIGN: A prospective randomised study in rats. METHODS: A human ovarian cancer cell line (IGR-OV1) was xenografted in nude rats. Seven days after cancer inoculation, surgery was performed. Rats were randomised in two main groups and underwent either: gasless laparoscopy (n = 20) CO2 laparoscopy with pneumoperitoneum pressure at 4 mmHg (n = 20), or 8 mmHg (n = 20) with in each case, increasing operative duration: 30,60,90 or 120 minutes (five rats for each time). Animals were killed seven days after the intervention. MAIN OUTCOME MEASURES: Tumour dissemination and frequency of port-site metastases. RESULTS: Tumour dissemination was not influenced by gas pressures or duration of procedure. The rate of rats with at least one port-site metastasis (one or two) was similar in all groups: gasless: n = 10/20; 4 mmHg CO2: 5/20; 8 mmHg CO2: 7/20,(P = 0.26). The number of port-site metastases were significantly higher in the gasless group compared with the 4 mmHg CO2 group (15/40 (37.5%) vs 5/40 (12.5%), P = 0.01). Difference was not significant between the 8mmHg group and the gasless group (9/40(22.5%) vs 15/40(37.5%), P = 0.14) or the 4mmHg group (9/40(37.5%) vs 5/40 (12.5%), P = 0.24). Duration of procedures had no significant influence on port-site metastases rate (P > 0.05). CONCLUSIONS: Unlike previous animal studies, port-site metastases were more frequent with gasless laparoscopy than with CO2 pneumoperitoneum. Local peritoneal factors could play an important role in port-site metastases mechanism.

Effect of pneumoperitoneum on circulating tumor DNA.

BACKGROUND: Release and circulation of tumor DNA could be favored by surgery. No data is available for the effect of laparoscopy on this phenomenon. MATERIAL AND METHODS: The aim of this study was to assess the impact of CO2 laparoscopy on circulating tumor DNA. Two xenografts of ovarian cancer were obtained by intraperitoneal inoculation (IP) of IGR-OV1 or NIH:OVCAR-3 cells in nude rats. CO2 laparoscopy (L), gasless laparoscopy (GL), midline laparotomy (ML) or general anesthesia as a control (C) were randomly carried out when the tumor graft was present in the peritoneal cavity. A sterile blood sample was taken in each case as soon as the experiment was completed. DNA was subsequently extracted and amplified (PCR, primers HLA GH 26 and HLA GH 27 specific for human DNA). In each model, we compared the influence of each surgical approach on circulating tumor DNA. Statistics were performed with the Wilcoxon test and Fisher exact test. 1: RESULTS: Eighteen rats were included in each group. Our protocol could detect an amount of tumor DNA equivalent to 10 cells/ml of blood. This technique was specific. Circulating tumor DNA was frequently observed in the IGR-OV1 model (45 to 50%), without significant difference between groups (p=0.6). In the NIH: OVCAR-3 model, the detection rate ranged from 22% (control group) to 64% (gasless group); but the overall comparison between the four groups was not significant (p = 0.2). CONCLUSION: In this experimental trial, CO2 laparoscopy had no deleterious effect on circulating tumor DNA. Biologic characteristics of tumors could also play a role.

Impact of pneumoperitoneum on visceral metastasis rate and survival. Results in two ovarian cancer models in rats.

OBJECTIVE: To compare the impact of CO2 laparoscopy, gasless laparoscopy, and midline laparotomy on the development of distant metastases and on survival in two ovarian carcinoma models. DESIGN: A prospective randomised study in rats. MATERIAL: and methods Two ovarian cancer xenografts were obtained by intraperitoneal injection of IGR-OV1 or NIH-OVCAR-3 cells. Experimental surgical procedures were performed on day 7 (IGR-OVI model) or day 14 (NIH: OVCAR-3 model) after intraperitoneal injection: CO2 laparoscopy (pneumoperitoneum (PNP) with unheated CO2 at a pressure of 8 mmHg for 1 hour); gasless laparoscopy (consisting in abdominal wall expansion by a balloon for 1 hour); midline laparoscopy (consisting in bowel exteriorisation on a mesh for one hour following xyphopubic laparotomy). The control group underwent general anaesthesia alone. The animals were killed by CO2 inhalation as soon as they became moribund. MAIN OUTCOME MEASURES: Pathological examination was carried out on the liver, lungs and pleura as well as the retroperitoneal nodes. Survival was determined from the time of surgery to the sacrifice of the animal. Statistical analysis used ANOVA, Fisher exact test, Bonferonni method and the log-rank test. RESULTS: In the IGR-OV1 model, distant metastases were rare, and were not promoted by CO2 laparoscopy. With the NIH: OVCAR-3 model, pleural, pulmonary and para-aortic metastases were not enhanced by CO2 PNP when compared with other approaches. Conversely, midline laparotomy and laparoscopy significantly increased liver involvement when compared with gasless laparoscopy (P = 0.04 and P = 0.008). Survival was comparable no matter what kind of surgery had been performed in the IGR-OV1 model (P = 0.7) or in the NIH: OVCAR-3 model (P = 0.5). CONCLUSIONS: CO2 laparoscopy had a minor impact on distant and nodal metastases in the two models. Similarly, survival was similar for all surgical groups.

Delineation of the insulin-responsive sequence in the rat cytosolic aspartate aminotransferase gene: binding sites for hepatocyte nuclear factor-3 and nuclear factor I.

Expression of the rat cytosolic aspartate aminotransferase gene is stimulated by glucocorticoids and repressed by insulin in the liver. The regulation by insulin and part of the glucocorticoid effect are mediated by a distal region in the promoter. A 142 bp fragment (-1844 to -1702) confers hormonal sensitivity to the heterologous thymidine kinase promoter in transient-transfection assays in H4IIEC3 hepatoma cells. Footprinting and gel-shift assays showed that several nuclear proteins bind to this region at conserved CCAAT-enhancer binding protein (C/EBP), activator protein (AP-1) and E-box sequences. Hepatocyte nuclear factor-3alpha (HNF-3)alpha and beta bind to sequences upstream of a glucocorticoid-responsive element (GRE) half-site as demonstrated by supershift experiments. Nuclear factor I (NFI)-like proteins bind downstream of the GRE half-site. These sites around the GRE motif overlap with five insulin responsive element (IRE) -like sequences (TG/ATTT). The effect of insulin was not prevented by any single mutation in the IRE-like sites. However, mutation of two IRE sites (namely IREc and d) prevented the insulin effect although only marginally affecting the glucocorticoid effect. The results suggest that the effect of insulin is due to a complex interplay of factors requiring the synergistic contribution of at least two sites and underline the contribution of HNF-3 and NFI-like proteins.

Identification of an adipocyte-specific negative glucose response region in the cytosolic aspartate aminotransferase gene.

Cytosolic aspartate aminotransferase (cAspAT) participates in gluconeogenesis in the liver and is expected to exert a glyceroneogenic function in the adipose tissue when the supply of glucose is limited. Here we demonstrate that adipose cAspAT messenger RNA (mRNA) is increased when rats are fed a low carbohydrate diet. In the 3T3-F442A, BFC-1 adipocyte cell lines and differentiated adipocytes in primary culture, a 24 h glucose deprivation induces approximately a 4-fold increase in cytosolic AspAT (cAspAT) mRNA, whereas mitochondrial AspAT mRNA remains unchanged. cAspAT activity is also increased in a weaker but reproducible manner. Addition of glucose within a physiological range of concentrations reverses the increase of cAspAT mRNA in 8 h (EC50 = 1.25 g/liter). Such a regulation requires protein synthesis and is specific for adipocytes differentiated in culture. It does not occur in Fao or H4IIE hepatoma cells, in C2 muscle cells, or in 293 kidney cells. 2-deoxyglucose mimicks glucose, while 3-orthomethyl-glucose has no effect, suggesting that glucose-6-phosphate is the effector. cAspAT mRNA stability is not affected by glucose deprivation. To ascertain the transcriptional nature of the glucose effect, we have stably transfected 3T3-F442A adipoblasts with constructs containing the chloramphenicol acetyltransferase reporter gene under the control of either 5'-deletions of the cAspAT gene promoter or internal fragments in an heterologous context. We demonstrate that a glucose response element(s) is present in the region between -1838 and -1702 bp relative to the translation start site. In this region, three DNA sequences bind nuclear proteins from adipocytes as shown by footprinting experiments. Our results indicate that cAspAT gene transcription is repressed by glucose selectively in adipocytes.

Fenofibrate modifies transaminase gene expression via a peroxisome proliferator activated receptor alpha-dependent pathway.

Fibrates modify the expression of genes implicated in lipoprotein and fatty acid metabolism via the peroxisome proliferator-activated receptor alpha(PPARalpha), leading to reductions in serum triglycerides and cholesterol. The expression of certain genes regulated by PPARalpha have been shown to be modified in a species dependent manner. Aspartate aminotransferase (AspAT or GOT) and alanine aminotransferase (AlaAT or GPT) are enzymes involved in intermediate metabolism in all cells and in hepatic gluconeogenesis. These enzymes are also widely used as serum markers of possible tissue damage. This study investigated whether fenofibrate could modify the expression of liver AspAT and/or AlaAT and thus possibly alter transaminase levels independently of a cytotoxic effect. In human Hep G2 cells, fenofibrate increased cytosolic AspAT (cAspAT) activity by 40% and AlaAT activity by 100%, as well as both mRNAs. Nuclear run on assays showed that this effect was, at least in part, transcriptional. Increases in mRNA were also observed in human hepatocyte cultures at concentrations of the drug attained in patients. In C57BL/6 mice, fenofibrate decreased cAspAT and cAlaAT mRNA, while these effects were abolished in PPARalpha knock-out mice. In conclusion, fenofibrate has been shown to modify cAspAT and AlaAT gene expression in a species and PPARalpha dependent manner. This is the first demonstration that cAspAT and AlaAT activities may be pharmacologically altered, independently of a toxic phenomenon.

Cytosolic aspartate aminotransferase gene is a member of the glucose-regulated protein gene family in adipocytes.

Stress controls the expression of a cohort of genes. Among these, the glucose-regulated protein (GRP) genes are specifically activated by glucose deprivation, reducing agents, glycosylation block, intracellular calcium or ex vivo incubations of tissues or cells. We demonstrate that these stimuli induce the expression of the cytosolic aspartate aminotransferase gene in adipocytes by a process involving the region of the promoter between -2405 and -26 bp. Therefore this transaminase is a new member of the GRP family.

Cell-specific, promoter-dependent mineralocorticoid agonist activity of spironolactone.

The agonist activity of the antimineralocorticoid spironolactone was evaluated in various cell lines through the use of transfection experiments. The target promoters were derived from the deltaMTV promoter in which one or several glucocorticoid-responsive elements (GRE) were inserted in tandem. Spironolactone at 100 nM activated by 6-fold the GRE/deltaMTV promoter in the human hepatoma HepG2 cell line and only partially prevented the 10-fold activation of this promoter by 0.1 nM aldosterone. Both effects were completely dependent on the cotransfection of an expression vector for the mineralocorticoid receptor. The half-maximal agonist effect of spironolactone was similar to its half-maximal antagonist effect (approximately 10 nM). For the GRE-2/deltaMTV, GRE-4/deltaMTV, and wild-type MMTV promoters, the activation by aldosterone was much more potent (70-, 100-, and 110-fold, respectively), whereas spironolactone elicited a 10-, 24-, and 25-fold activation, respectively. Thus, the effect of both compounds and the relative efficiency of spironolactone, compared with that of aldosterone, were dependent on the number of GREs present in the regulatory region of the promoter. The agonist effect of spironolactone was cell specific. Indeed, although spironolactone agonist activity was observed in H5 kidney tubule cells, none could be detected at concentrations of < or = 1 microM in the CV1 monkey fibroblast cells. In contrast, the antagonist effect was observed in all cells. Furthermore, other antimineralocorticoids, such as RU 26752 and progesterone, also displayed mineralocorticoid receptor-dependent agonist activity in the HepG2 cells. The antiprogesterone RU 486 and the antiandrogen cyproterone acetate were ineffective at < or = 1 microM. In conclusion, we show that under certain experimental conditions, several antimineralocorticoids display significant agonist activity in a cell-specific and promoter-dependent manner.

Contribution of a nuclear factor 1 binding site to the glucocorticoid regulation of the cytosolic aspartate aminotransferase gene promoter.

Two regions of the cAspAT gene promoter mediate the glucocorticoid regulation of this gene in the Fao hepatoma cell line. The proximal region was localized by deletion studies and stable transfections in the Fao cells to the sequence -553/-398. This region includes the glucocorticoid-responsive element (GRE) A sequence, which consists of two overlapping GREs and which can mediate the glucocorticoid regulation of a heterologous promoter. DNase I footprinting studies have shown that a site 80 base pairs upstream of the GRE A was protected by liver and brain nuclear extracts (site P8). The binding was displaced by an excess of an oligonucleotide containing a typical NF1 binding site and by NF1-specific antibodies. In electrophoretic mobility shift assay using the P8 oligonucleotide as a probe, several complexes were formed. Most complexes were common to liver and brain but were less abundant when testis extracts were used. At least one complex was specific to the liver. All complexes, with the exception of two, were competed for by the NF1 oligonucleotide. Furthermore, the sequence of the P8 site showed a 7/9-base pair homology with a typical NF1 site. A mutation of the P8 site, which prevents the binding of NF1-like proteins to it, considerably decreases the regulation of the cAspAT promoter fragment by glucocorticoids. Surprisingly, the basal activity of the mutant promoter was increased 2-fold. Thus, the regulation of the cAspAT gene promoter is mediated by a regulatory unit comprising the GRE A and a NF1 binding site.

Acinar zonation of the hormonal regulation of cytosolic aspartate aminotransferase in the liver.

The zonation of the expression and regulation of the cytosolic aspartate aminotransferase (cAspAT) mRNAs in the liver acinus was investigated in diabetic and/or adrenalectomized rats. Dexamethasone increased cAspAT activity two- to threefold alone and up to sixfold in combination with streptozotocin-induced diabetes. Northern blot analysis showed that the cAspAT mRNAs were increased by those treatments; the effect of streptozotocin was reversed by the administration of insulin. In situ hybridization experiments showed that basal cAspAT mRNAs were uniformly distributed within the liver acinus. However, cAspAT mRNAs were induced by glucocorticoids specifically in the periportal zone and by streptozotocin in a larger area including the periportal and intermediary zone. The alpha 2u-globulin mRNAs which are specifically expressed in the perivenous hepatocytes are also induced by glucocorticoids in this zone, suggesting that the specific regulation of the cAspAT gene by glucocorticoids in the periportal zone is not due to the absence of functional glucocorticoid receptors in the other zones. We conclude that the regulation of the cAspAT housekeeping gene is zone specific in the liver. Furthermore, this zonation depends on the gene and on the type of hormonal or pharmacological treatment.

Phorbol esters inhibit the glucocorticoid-mediated stimulation of cytosolic aspartate aminotransferase gene transcription.

The regulation of cytosolic aspartate aminotransferase (cAspAT) gene expression by phorbol esters was investigated in the highly differentiated hepatoma cell line Fao. Phorbol 12,13-dibutyrate (PdBu) had no effect on basal activity but partially inhibited the induction of cAspAT by dexamethasone. The extent of inhibition (40%) was similar to that obtained with insulin or vanadate. The inhibitory effects of PdBu and vanadate were additive. In the case of PdBu, the inhibitory effects could be eliminated by first incubating the cells with PdBu, which down-regulates protein kinase C. In contrast, inhibition by insulin was not modified by this treatment. The molecular mechanism of PdBu action was investigated. Northern blot analysis showed that the steady-state mRNA levels of cAspAT were decreased by PdBu in the presence of dexamethasone. In addition, the transcription rate, as measured by run-on experiments, was also decreased under the same conditions. Finally, a 2.4 kb promoter fragment driving the chloramphenicol acetyltransferase gene was stably transfected into the Fao cells. The regulation of the activity of this promoter fragment by dexamethasone and PdBu was similar to the regulation of the endogenous cAspAT activity. We conclude that PdBu acts by regulating the promoter activity of the cAsPAT gene.

Cell-specific regulation of cytosolic aspartate aminotransferase by glucocorticoids in the rat kidney.

The basal expression and hormonal regulation of cytosolic aspartate aminotransferase (cAspAT) were investigated in the rat kidney. In adrenalectomized animals, the basal activity was highest in the renal cortex and in the inner stripe of the outer medulla (0.1-0.15 U/mg protein). The glucocorticoid analogue dexamethasone increased cAspAT activity about twofold in the cortex and in the inner stripe of the outer medulla but not in the papilla. A half-maximal increase in the activity was achieved at doses of approximately 5 micrograms/100 g body wt. The mineralocorticoid aldosterone did not modify the cAspAT activity. The cell specificity of the hormonal regulation was analyzed by in situ hybridization. In untreated adrenalectomized rats, a cAspAT cRNA probe labeled mainly the inner stripe of the outer medulla. After dexamethasone or hydrocortisone treatment, labeling was uniformly increased in this part of the medulla and was heterogeneously increased in the renal cortex. The specific increase in labeling within the cortex was shown to be confined to the distal convoluted tubule and the thick ascending limb. We conclude that, in addition to widespread basal expression, cAspAT is regulated by glucocorticoids in a highly cell-specific manner in the renal cortex. The enzyme may thus participate in the increased energy metabolism elicited by these hormones in these cells.

Regulation of the cytosolic aspartate aminotransferase housekeeping gene promoter by glucocorticoids, cAMP, and insulin.

The cytosolic aspartate aminotransferase (cAspAT) is a ubiquitous enzyme that displays liver-specific hormonal regulation. In the hepatoma cell line Fao, both the activity and the mRNA level of cAspAT are increased by glucocorticoids. This effect is potentiated by cAMP and inhibited by insulin. Using in vivo run-on experiments, we showed that these effectors act at the transcriptional level. A cAspAT gene fragment containing 2405 bp of the promoter was sequenced. Deletion fragments of this promoter were inserted upstream of the CAT gene, and the regulation of their activity was assayed following transfection in Fao cells. Stable transfection experiments established that the construct including the entire 2.405-kb fragment undergoes positive regulation by glucocorticoids and cAMP and negative regulation by insulin similar to the regulation of the endogenous gene. A physical separation of the positive and negative control elements is suggested by the fact that cAMP acted on the -682/-26-bp fragment (a 2-fold increase of the stimulation by dexamethasone), whereas the negative regulation by insulin (50% of the stimulation by dexamethasone) required the -1983/-1718-bp fragment. Both regions were required for maximal glucocorticoid activity (6-9-fold increase of CAT activity). We conclude that at least two regulatory regions, a proximal and a distal one, are required for full hormonal regulation of the cAspAT gene.

CCAAT/enhancer-binding protein-related proteins bind to the unusual promoter of the aspartate aminotransferase housekeeping gene.

The cytosolic aspartate aminotransferase (cAspAT) gene is ubiquitously expressed but it is regulated by hormones in a tissue-specific manner. In vitro DNase I footprinting studies of a 260-base pair fragment carrying the basal promoter activity revealed that three CCAAT sequences bind liver nuclear proteins (protected regions P2, P3, P4). Competition studies, the heat resistance of these proteins, and identical footprints obtained using a recombinant CCAAT/enhancer-binding protein (C/EBP) alpha fragment indicate that they belong to the C/EBP family of transcription factors. A fourth protected region P1, overlapping the P2 region, was observed in the liver in the presence of competing oligonucleotides containing the C/EBP site. In cotransfection experiments, the C/EBP beta protein trans-activated 10-15-fold the cAspAT gene promoter in HepG2 cells. Deletion studies revealed that regions P2 and P4 are critical for promoter activity. In gel retardation experiments, the P4 region bound different C/EBP-related proteins in different tissues: the brain protein is heat sensitive in contrast to the liver protein. The synthetic oligonucleotide OL 1-2, which covers the P1 and P2 regions, binds C/EBP-like proteins as well as NF1 and CP1 (or NFY) transcription factors, but the preferential binding of one of these protein is tissue-specific. In summary, using the cAspAT gene promoter, we have shown that ubiquitously active promoters may be recognized by different proteins in different tissues.

Organization of the 5' end of the rat gamma-glutamyl transpeptidase gene: structure of a promoter active in the kidney.

Two gamma-glutamyl transpeptidase mRNAs (mRNAI and mRNAII), with alternate 5'-untranslated regions, are expressed in the rat kidney. Oligonucleotides were designed based upon these two alternate 5' sequences and used as primers to amplify GGT genomic DNA sequences. The genomic organization of the mRNAI and mRNAII 5'-untranslated sequences reveals that the mRNAs are encoded from two separate promoters which are 2.1 kbp apart on the single GGT gene. A 2775 base pair genomic sequence, which contains the proximal GGT promoter, was cloned from two overlapping amplified fragments. S1 mapping analysis shows that the kidney GGT mRNAI is transcribed from several start sites on this promoter which displays neither a classical TATA box nor Sp1 binding sites. Chimeric plasmids, including the GGT promoter region for mRNAI, associated with the chloramphenicol acetyltransferase (CAT) reporter gene, were transiently expressed in a kidney (LLCPK) and in a hepatoma (HTC) cell line. A sequence extending 308 bases upstream from the major GGT mRNAI start site drives a promoter activity which is 5-fold higher in LLCPK than in HTC cells and is sufficient to confer cell specificity to the GGT proximal promoter.