Alpha-fetoprotein mRNA Levels Research Articles

Relationship between Protein kinase C isoforms, Telomerase and Alpha-fetoprotein through PI3K/AKT/mTOR pathway in Hepatocellular carcinoma

Protein kinase C (PKC) family has been an alluring objective for new cancer drug discovery. It has been reported to regulate telomerase in several cancer types. Our team had previously used telomerase to elucidate alpha-fetoprotein (AFP) modulation in hepatocellular carcinoma (HCC). The aim of this study was to investigate the interrelationships among PKC isoforms, telomerase and AFP in HCC. PKCα and PKCδ were the most expressed isoforms in HepG2/C3A, PLC/PRF/5, SNU-387 and SKOV-3 cells. Following the upregulation of AFP using pCMV3-AFP and the human telomerase reverse transcriptase (hTERT) using a construct expressing a wild-type hTERT, and after their inhibition with all-trans retinoic acid and hTERT siRNA each respectively, we found that the expression of PKCα, PKCβI, PKCβII and PKCδ was affected by the variation of AFP and hTERT mRNA levels. An increase in AFP expression and secretion was observed after gene silencing of PKCα, PKCβ, PKCδ, and PKCε in HepG2/C3A. A similar pattern was observed in transfected PLC/PRF/5 cells, however PKCδ isoform silencing decreased AFP expression. Furthermore, telomerase activity was quantified using quantitative telomeric repeat amplification protocol. The variations in hTERT expression and telomerase activity were similar to those of AFP. Further investigation showed that PKC isoforms regulate AFP and hTERT expression levels through PI3K/AKT/mTOR pathway in HepG2/C3A and PLC/PRF/5 cells. Thus, these results show for the first time a possible interrelationship that links PKC isoforms to both AFP and hTERT via PI3K/AKT/mTOR pathway in HCC.

TUG1 Is a Regulator of AFP and Serves as Prognostic Marker in Non-Hepatitis B Non-Hepatitis C Hepatocellular Carcinoma.

Thyroid hormone (T3) and its receptor (TR) are involved in cell metabolism and cancer progression. Hypothyroidism is associated with significantly elevated risk of hepatocellular carcinoma (HCC). Levels of the glycoprotein alpha-fetoprotein (AFP) are increased in the majority of patients with HCC and may be useful in diagnosis and follow-up. However, the relationship between T3/TR and AFP levels in HCC is currently unclear. The expression profiles of long non-coding RNAs (lncRNAs) were compared in microarrays of HepG2-TRα1 cells treated with/without T3 and HCC specimens. The effects of T3 on taurine upregulated gene 1 (TUG1) and AFP expression were validated using qRT-PCR. A correlation between TUG1 and AFP was confirmed via RNAi and clustered regularly interspaced short palindromic repeats (CRISPR) strategies. Finally, overall and recurrence-free survival rates were analyzed using the Kaplan–Meier method and confirmed in online datasets. T3/TR treatment reduced TUG1 expression in vitro, resulting in the downregulation of AFP mRNA. Knockdown of TUG1 suppressed cell cycle progression and soft agar colony formation and induced cellular senescence. Our data support the involvement of TUG1 in the T3/TR-mediated suppression of cell growth. AFP mRNA levels showed strong positive correlations with TUG1 and unfavorable prognosis in patients with non-hepatitis B/non-hepatitis C HCC (NBNC-HCC). T3/TR, TUG1, and AFP may potentially serve as effective prognostic markers for NBNC-HCC.

PO-041 TNF pathway in metastatic colorectal cancer according to RAS status and implication of potassium channels

IntroductionDespite its role as a diagnostic and prognosis marker for Hepatocellular carcinoma (HCC), alpha fetoprotein (AFP) plays a key role in advancing tumorigenesis and tumour expansion. Telomerase, an enzyme elongating telomere length, is upregulated in 80% of cancers including HCC. Our team had elucidated a modulation of AFP by telomerase and protein kinase C (PKC). PKC family is formed of several isoforms, each with a specific cellular function and expression. The aim of this study is to investigate the interrelation between PKC isoforms, telomerase and AFP in HCC.Material and methodsPKC isoforms were quantified by RT-qPCR in two AFP secretory cell lines, HepG2/C3A and PLC/PRF/5 and two non-secretory AFP cell lines SNU-387 (hTERT+) and SKOV-3 (hTERT-). According to the results, the expression of four isoforms was suppressed by si-RNAs in HepG2/C3A and PLC cells. AFP and telomerase mRNA levels were quantified in transfected cells by q-PCR, and AFP secretion by ELISA. Toxicity and cell proliferation were assessed by WST-1. In order to examine the effect of the dual presence of AFP and hTERT on PKC isoforms, SNU-387 and SKOV-3 were transfected with AFP expression plasmid pCMV3-AFP, then PKC isoforms mRNA was assessed by qPCR.Results and discussionsFour PKC isoforms, alpha, beta, delta and epsilon exhibited the highest expression levels in all cells compared to the remaining isoforms. An increase in telomerase expression, AFP expression and secretion and cell proliferation reaching a maximum of two folds was observed after individual gene silencing of the four isoforms in HepG2/C3A cells; however, an inverse pattern was noticed when using PKC pan inhibitor Go6983. Similar results were observed in PLC cell line. The expression of the four isoforms increased in SNU-387 and SKOV-3 cells after 24 hour transfection. The effect persisted after 48 hour in SNU-387, contrary to SKOV-3 where the levels decreased returning to the controls expression levels.ConclusionTaken together, decreased individual PKC isoforms rise telomerase expression and AFP secretion. However, PKC isoforms overexpression requires the presence of hTERT in AFP secretory cells. Thus, these results show for the first time the possible inter relation linking PKC isoforms to both AFP and hTERT in HCC.

AFP anti‐sense transcripts in mouse liver and their potential role in gene regulation

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, ranking the sixth most common cancer and third most common cause of cancer mortality worldwide. We have been studying mouse liver gene regulation to better understand mechanisms by which changes in gene expression contribute to liver development, homeostasis and disease. We identified Zinc Fingers and Homeoboxes 2 (Zhx2) as a regulator of alpha‐fetoprotein (AFP), a plasma protein that is highly expressed in the fetal liver but is shut off after birth. AFP expression is elevated in regenerating adult liver and hepatocellular carcinoma (HCC) and has been used extensively as a diagnostic marker of liver cancer. Interestingly, all genes that are misregulated in the absence of Zhx2 are also misregulated in HCC. Thus, to better understand gene regulation during HCC, we have been studying of the mechanism by which Zhx2 regulates gene expression. While studying AFP mRNA regulation by Zhx2, we identified previously unannotated antisense transcripts (asAFP) that partially overlap the 3′ half of the mouse AFP gene. asAFP RNAs are ~5kb alternatively spliced, mainly cytoplasmic, transcripts containing 2–4 exons that are likely to be non‐coding RNAs. These antisense transcripts were also detected in mouse liver RNA‐seq data. The abundance of asAFP RNA inversely correlates with AFP mRNA levels during postnatal development. Normally, asAFP RNA levels are high and AFP mRNA levels are low in the adult mouse liver. However, in the absence of Zhx2, AFP mRNA levels are higher and asAFP RNA levels are reduced. When portions of the asAFP RNA are over‐expressed in a liver cell line, endogenous AFP mRNA was reduced, suggesting asAFP transcripts repress AFP mRNA expression in trans. We will also knock‐down asAFP RNA and predict that AFP mRNA levels will increase. My central hypothesis is that asAFP RNA contributes to the post‐transcriptional regulation of AFP mRNA through an RNA‐RNA interaction. We are designing experiments to test this hypothesis and to determine at what level the regulation may occur.Support or Funding InformationMCB‐1158234 from the National Science Foundation, R01‐DK59866 from the National Institutes of Health

Zhx2 and the Regulation of AFP Anti‐sense RNAs in Mouse Liver

Although the genomes of eukaryotes are mostly transcribed, only a very small percentage of the transcripts code for proteins. The majority of the transcripts do not have known functions. In recent years, growing evidence has suggested that these non‐coding transcripts may play important roles in gene regulation. In the past two years, our lab has identified and characterized long non‐coding RNAs that are transcribed from the anti‐sense strand of, and partially overlap with, the alpha‐fetoprotein (AFP) gene. AFP is highly expressed in fetal liver, but its expression decreases to a very low level after birth. In adult liver, AFP expression increases during regeneration due to liver damage or during the onset of hepatocellular carcinoma. Our study has shown that anti‐sense AFP (asAFP) transcripts are alternatively spliced and their abundance is inversely correlated with AFP mRNA during liver development. While these asAFP RNAs had not been annotated, RNA‐seq data from mouse liver confirmed and extended our characterization and ENCODE data for histone modifications and DNase I hypersensitivity show signals near the asAFP exons we have identified. Expression levels of AFP mRNA and asAFP RNA also are inversely correlated in the presence or absence of the Zinc fingers and homeoboxes 2 (Zhx2) gene. Zhx2, a putative transcription factor, was shown previously to contribute to the developmental repression of AFP; in the absence of Zhx2, AFP mRNA is high while asAFP RNA is low, compared to wild‐type. Transiently transfecting asAFP into a liver cell line causes a decrease in endogenous AFP, showing asAFP contributes to AFP regulation in trans. We have shown that the asAFP transcripts localize mainly in the poly(A)+ fraction of cytoplasmic RNA, but this does not identify where the asAFP RNA functions. To determine whether the asAFP RNA represses AFP mRNA in the nucleus or cytoplasm, we have been analyzing the abundance of these two RNAs in nuclear and cytoplasmic RNA from liver of Zhx2 wild‐type or knock‐out animals. To better understand the role of Zhx2 in modulating AFP and asAFP RNA expression, future studies will use chromatin IP to determine whether Zhx2 interacts directly with these genes. To identify which of the genes that are mis‐regulated in the absence of Zhx2 are direct targets, we plan to do Zhx2 ChIP‐seq experiments.Support or Funding InformationMCB‐1158234 from the National Science Foundation, R01‐DK59866 from the National Institute of Health

Constitutive Notch2 signaling induces hepatic tumors in mice

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCC) are the most common liver tumors and a leading cause for cancer-related death in men. Notch2 regulates cellular differentiation in the developing and adult liver. Although aberrant Notch signaling is implicated in various cancers, it is still unclear whether Notch2 regulates proliferation and differentiation in liver carcinogenesis and thereby contributes to HCC and CCC formation. Here, we investigated the oncogenic potential of constitutive Notch2 signaling in the liver. We show that liver-specific expression of the intracellular domain of Notch2 (N2ICD) in mice is sufficient to induce HCC formation and biliary hyperplasia. Specifically, constitutive N2ICD signaling in the liver leads to up-regulation of pro-proliferative genes and proliferation of hepatocytes and biliary epithelial cells (BECs). Using the diethylnitrosamine (DEN) HCC carcinogenesis model, we further show that constitutive Notch2 signaling accelerates DEN-induced HCC formation. DEN-induced HCCs with constitutive Notch2 signaling (DEN(N2ICD) HCCs) exhibit a marked increase in size, proliferation, and expression of pro-proliferative genes when compared with HCCs from DEN-induced control mice (DEN(ctrl) HCCs). Moreover, DEN(N2ICD) HCCs exhibit increased Sox9 messenger RNA (mRNA) levels and reduced Albumin and Alpha-fetoprotein mRNA levels, indicating that they are less differentiated than DEN(ctrl) HCCs. Additionally, DEN(N2ICD) mice develop large hepatic cysts, dysplasia of the biliary epithelium, and eventually CCC. CCC formation in patients and DEN(N2ICD) mice is accompanied by re-expression of hepatocyte nuclear factor 4α(HNF4α), possibly indicating dedifferentiation of BECs. Our data establish an oncogenic role for constitutive Notch2 signaling in liver cancer development.

Abstract 2415: TGF-ß signaling inactivation inhibits the formation of hepatocellular carcinomas induced by loss of p53

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most deadly forms of cancer and results from the accumulation of mutated or altered tumor suppressor genes and/or oncogenes. The tumor suppressor and oncogenes commonly affected include growth factors, receptors and downstream signaling components. Inactivation of the TGF-ß signaling pathway and mutation of TP53, the gene for p53, are common in HCCs suggesting they may cooperate in HCC formation. Thus, we assessed whether inactivation of TGF-ß signaling, by deletion of the TGF-ß receptor type II, TGFBR2, affects the formation of HCCs arising secondary to loss of p53. Aim: To determine if loss of TGFBR2 and p53 cooperate in vivo to affect HCC formation. Methods and Results: Albumin-cre (Alb-Cre) transgenic mice were crossed with mice homozygous for floxed Tgfbr2 (Tgfbr2flx/flx) and/or floxed Trp53 (Trp53flx/flx) to generate mice lacking TGFBR2 and/or p53 in the liver. Deletion of Tgfbr2 alone did not induce liver tumors, while inactivation of both Tgfbr2 and Trp53 resulted in a subset of mice (19%) that developed liver tumors by 65 weeks of age. Surprisingly, deletion of p53 alone, in the context of intact TGF-ß receptors, resulted in an increased number of mice developing tumors (38%), as well as increased morbidity (42 weeks of age). Interestingly, in both genotypes, Alb-Cre;Trp53flx/flx;Tgfbr2flx/flx and Alb-Cre;Trp53flx/flx;Tgfbr2wt/wt, the mice develop both hepatocellular carcinomas (HCC) and cholangiocarcinomas (CC), suggesting these tumors originate from a common liver stem cell population. Quantitative RT-PCR analysis of tumor and non-tumor tissue demonstrated a subset of Alb-Cre;Trp53flx/flx;Tgfbr2wt/wt (p53 null) tumors express significantly increased levels of alpha-fetoprotein mRNA, a clinical marker for HCC. Additionally, tumors from the Alb-Cre;Trp53flx/flx;Tgfbr2wt/wt displayed increased TGFβ-1 protein levels as compared to tumors from Trp53 and Tgfbr2 null mice. Furthermore, increased phosphorylated ERK1/2 expression was also present in the tumors from the p53 null mice and correlated with a slight up-regulation in beta-catenin expression, another common molecular event observed in human HCC. Conclusions: The Alb-Cre;Trp53flx/flx;Tgfbr2flx/flx mouse model recapitulates many molecular features of human HCC and demonstrates that TGF-ß signaling is oncogenic in the setting of loss of p53 in the liver. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2415. doi:10.1158/1538-7445.AM2011-2415

Postnatal AFP Repression by Zhx2 through RNA Splicing Inhibition

The alpha‐fetoprotein (AFP) gene is highly expressed in the fetal liver and repressed at birth. This postnatal repression is controlled, in part, by Zhx2 (PNAS, 102:396). BALB/cJ mice have reduced Zhx2 expression and higher adult liver steady‐state AFP mRNA levels compared to other mouse strains. The AFP promoter is sufficient to confer Zhx2‐mediated regulation to a reporter gene in transgenic mice, suggesting that the promoter is required for repression. Despite differences in steady state AFP mRNA levels in the adult liver between BALB/cJ and wild‐type mice, transcription rates across this gene were similar. We found accumulated unspliced RNA across multiple AFP introns in wild‐type mice where mature AFP mRNA levels are low. In our analysis of mRNA from cytoplasmic and nuclear RNA fractions of BALB/cJ mouse liver with or without the Zhx2 transgene, we found unprocessed cytoplasmic AFP transcripts in the presence of Zhx2, consistent with a global repression of splicing. We are purifying Zhx2‐associated proteins from liver nuclear extracts; the identity of co‐purified factors will provide insight into the mechanism by which Zhx2 couples transcriptional/post‐transcriptional control of AFP repression.

Alpha-fetoprotein and human telomerase reverse transcriptase mRNA levels in peripheral blood of patients with hepatocellular carcinoma

Tumor-associated gene expression in peripheral blood reflects the presence of circulating hepatocellular carcinoma (HCC) cells and might be associated with aggressive features of HCC. We assessed the prognostic significance of alpha-fetoprotein (AFP) and human telomerase reverse transcriptase protein (hTERT) mRNA expression in the peripheral blood of HCC patients. About 343 HCC patients, treated at the National Cancer Center, Korea, were included in this study. We measured AFP and hTERT mRNA levels in peripheral blood using quantitative real-time reverse transcription polymerase chain reaction. The association between the expression of each gene and survival was analyzed. AFP and hTERT mRNA were detected in 204 (59.5%) and 48 (14.0%) patients with HCC, respectively. The mean AFP copy number was 203 copies/microL (range 0-19992 copies/microL) and that of hTERT was 26 copies/microL (0-1711 copies/microL). AFP mRNA expression was correlated with the number of tumors (P = 0.05), but there were no significant association between hTERT expression and clinical features. There was also no relationship between overall survival and AFP (P = 0.08) or hTERT (P = 0.67) mRNA expression. This is the first report to evaluate the association between peripheral blood hTERT mRNA expression, and survival of HCC patients. The results indicate that AFP and hTERT mRNA expression in peripheral blood may not be useful HCC prognostic markers.

Expression and location of α-fetoprotein during rat colon development

To investigate the expression of alpha-fetoprotein (AFP), a cancer-associated fetal glycoprotein, and its involvement during rat colon development. Colons from Sprague-Dawley rat fetuses, young and adult (8 wk old) animals were used in this study. Expression levels of AFP in colons of different development stage were detected by reverse-transcriptase PCR (RT-PCR) and Western blotting. To identify the cell location of AFP in the developing rat colons, double-immunofluorescent staining was performed using antibodies to specific cell markers and AFP, respectively. The highest levels of AFP mRNA were detected in colons of rats at embryonic day 18.5 (e18.5). Compared to e18.5 d, the AFP expression was significantly decreased during rat development [85% for e20.5, P < 0.05, 58% for postnatal day 0.5 (P0.5), P < 0.05, 37% for P7, P < 0.05, 24% for P14, P < 0.05, and 11% for P21, P < 0.05] and undetected in adult rats. Only the 72-kDa isoform of AFP was detected by Western blotting, the expression pattern was similar to AFP mRNA and conformed to the results of mRNA expression. The AFP positive staining was identical to different distribution patterns in fetuses, young and adult animals and positive staining for both AFP and vimentin was overlapped in mesenchymal cells at each stage tested. This study has for the first time demonstrated that AFP is localized in the mesenchyme of rat colon from the embryo to the weaning stage by immunofluorescence and presents 72-kDa isoform in the developing rat colons by Western blotting. The dynamic expression of AFP in the various developmental stages of the colon indicates that AFP might be involved in many aspects of colon development.

Increased steady-state levels of alpha-fetoprotein mRNA in hepatocellular carcinoma: an analysis by in situ hybridization.

The molecular basis for the augmented production of alpha-fetoprotein is unknown. We have used in situ hybridization of alpha-fetoprotein cDNA to malignant hepatocytes to establish if increased serum alpha-fetoprotein concentrations are related to detectable steady-state levels of alpha-fetoprotein mRNA in hepatocytes. Tumor tissue from four patients with histologically confirmed hepatocellular carcinoma were examined, and the results compared to fetal liver. Northern blot hybridization for alpha-fetoprotein mRNA in tumor tissue was also analyzed. As expected a high number of grains was observed in fetal liver tissue, indicative of high levels of alpha-fetoprotein mRNA physiologically present during pre-natal development. Sections from all patients with high serum concentrations of alpha fetoprotein showed appreciable hybrid formation, which correlated semi-quantitatively with the serum concentrations. However, hybrids were not detected in a patient with a normal serum alpha-fetoprotein. The high alpha-fetoprotein mRNA levels in fetal and neoplastic liver suggest that gene transcription is the mechanism of alpha-fetoprotein production in malignancy, although the control of this mechanism remains speculative.

Analysis of Alpha-fetoprotein mRNA Level on the Tumor Cell Hematogenous Spread of Patients With Hepatocellular Carcinoma Undergoing Orthotopic Liver Transplantation

The aim of this study was to detect alpha-fetoprotein (AFP) mRNA levels in the preoperative and intraoperative peripheral blood of patients with hepatocellular carcinoma (HCC) receiving orthotopic liver transplantation (OLT). Methods We detected AFP mRNA by TaqMan real-time reverse transcriptase-polymerase chain reactions (RT-PCR) on the peripheral blood cells (PBCs) from 30 HCC patients undergoing OLT. Results In RT-PCR, fluorescence was undetectable in any control. The positive expression rate of AFP mRNA was 23% (7 of 30) in PBC samples of OLT patients preoperatively and 50% (15 of 30) just before hepatectomy during the operation. The positive rate of AFP mRNA in OLT patients at this time was higher than that at preoperation, a difference that was statistically significant ( P < .01). Conclusion The OLT operation induced release of cells from the liver into the peripheral blood circulation. This may be an important mechanism of liver cancer cell dissemination deserving further investigation.

Quantitation of alpha-fetoprotein messenger RNA for early detection of recurrent hepatocellular carcinoma: A prospective pilot study

Background: Alpha-fetoprotein (AFP) messenger RNA (mRNA) may be a potential marker of the dissemination of hepatocellular carcinoma (HCC) cells into the circulation. The aim of this prospective pilot study was to assess the prognostic value of quantitative levels of AFP mRNA in patients undergoing ablative treatment for HCC. Methods: Peripheral blood samples were taken from seven patients before and after treatment for measurement of AFP mRNA levels by reverse-transcriptase polymerase chain reaction (RT-PCR). Patients were treated with percutaneous radiofrequency thermal ablation ( n = 3) or transarterial chemoembolization ( n = 4). The level of AFP mRNA in blood was serially determined, and the time course was related to the clinical course and disease outcome. The median duration of follow-up was 14 months (range, 9–16 months). Results: HCC recurred locally in four patients, and lung metastases developed in two of them. Patients were divided into three groups on the basis of the pre- and post-treatment AFP mRNA status. Group 1 included four patients with consistently high serum AFP and AFP mRNA levels (pre- and post-treatment). These patients developed distant and local recurrence. Group 2 included a patient with serum-negative AFP mRNA and normal AFP levels at entry. Although serum AFP remained within normal range, mean AFP mRNA increased from 10 to 95 copies/μg RNA. This patient had no distant metastases, but his tumor markedly increased in size. In Group 3, AFP mRNA and serum AFP remained within normal range before and after treatment. These two patients did not develop either local or distant metastases during the follow-up period. Conclusions: Although this is a small sample size pilot study these findings imply that quantitative measurement of AFP-expressing cells in peripheral blood may serve as a marker of HCC recurrence.

Association Between Recurrence of Hepatocellular Carcinoma and α-Fetoprotein Messenger RNA Levels in Peripheral Blood

Intra- and extrahepatic recurrence is common, even after curative resection for hepatocellular carcinoma (HCC), suggesting preoperative or intraoperative tumor cell dissemination. Reverse transcription - polymerase chain reaction (RT-PCR) for alpha-fetoprotein (AFP) is used to detect circulating liver cancer cells. We previously developed a quantitative method that allows estimation of the AFP mRNA level by real-time PCR. In the present study, we used this method to measure the AFP mRNA level before and after resection of HCC, then correlated the findings with various clinicopathological characteristics and prognosis. We prospectively examined peripheral blood samples from 38 patients with HCC, and bone marrow aspirate from 25 of these patients. As a control, we examined bone marrow from 20 patients with benign diseases. The follow-up period ranged from 32 to 66 months. Real-time RT-PCR was used to detect AFP mRNA levels in the samples. AFP was expressed in 9 (23.7%) of the 38 peripheral blood samples. The detection of AFP mRNA was significantly correlated with extrahepatic metastasis after primary surgery, and a shorter disease-free survival time (P = 0.0245 each). Bone marrow samples were defined as positive if they expressed AFP mRNA at levels higher than the maximum expressed level in the controls, because only 1 (5%) of the 20 control bone marrow samples had low AFP mRNA expression. Using this cutoff level, 12 (48%) of the 25 patients with HCC had positivity for AFP mRNA. The results of bone marrow RT-PCR did not correlate with the clinocopathological characteristics of prognosis. Using real-time PCR to measure the AFP mRNA level in blood, but not bone marrow, could be useful for predicting postoperative tumor recurrence.

Simple quantitative assay of alpha-fetoprotein mRNA in liver tissue using the real-time detection polymerase chain reaction assay — its application for clinical use

Alpha-feto protein (AFP) mRNA levels increase in hepatocellular carcinoma (HCC) cells as compared with non-neoplastic tissue. Therefore, detection of AFP mRNA in blood nuclear cells is useful for the evaluation of treatment efficacy and prognosis of HCC. In this study, simple and reproducible methods were developed to quantify AFP mRNA using the real-time RT-PCR assay (Taq Man assay). By using in vitro synthesized AFP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA, the sensitivity and dynamic range of the RT-PCR assay were established. AFP mRNA in both HCC and non-neoplastic tissue, as well as in cell lines, were measured using this assay system. The expression of the AFP mRNA level was normalized using the GAPDH house keeping gene product as an endogenous reference. AFP and GAPDH mRNA can be quantified in the range of 10–10 8 copies when using this quantitative assay. Among HCC cell lines, Huh 7 and HepG2 cells, respectively, represented 1.5×10 6 and 6.0×10 5 AFP mRNA/10 6 GAPDH mRNA, in contrast to 6, 23 and 230 AFP mRNA/10 6 GAPDH mRNA for HLE, HLF and PLC/PRF/5 cells, respectively. Other cell lines derived from stomach, pancreas, and colon cancers have 10 AFP mRNA copies/10 6 GAPDH mRNA. In liver tissue from patients with chronic hepatitis, and the non-neoplastic portion of the liver from HCC patients, AFP mRNA distributes from 2.5×10 3 to 5.8×10 4/10 6 GAPDH transcripts. In contrast, AFP mRNA in tumor cells were more than 100-fold higher than that found in corresponding non-neoplastic portions in two patients who had a high level of AFP in serum. The establishment of the TaqMan quantifying system for AFP mRNA may have important clinical implications.

Rat yolk sac explants as a system for studying the regulation of endodermal genes: Down-regulation of the alpha-fetoprotein gene by clexamethasone and phorbol ester

The visceral yolk sac is a fetal membrane with essential placental functions. It is the major site of synthesis of alpha-fetoprotein (AFP), the most abundant plasma protein in the fetus. We developed a system of rat yolk sac explants in serum-free culture medium to study the regulation of endodermal gene expression in yolk sac. The explanted yolk sac tissues retained their double-sided morphology for up to 48 hours. The epithelial cells of both layers remained tightly joined on a basement membrane as seen by light and electron microscopy. This probably accounts for the continued expression of several endodermal cell-specific markers. The levels of mRNA encoding AFP, vitamin D-binding protein (DBP), hepatocyte nuclear factor 1alpha and beta transcription factors did not change during the 48-hour culture period. This reflects the stability of the differentiation state of the yolk sac endodermal cells. Dexamethasone and phorbol ester (TPA) specifically reduced the AFP mRNA level without affecting that of DBP. This suggests that these transduction pathways are functional in the yolk sac during this period of gestation and could be involved in the physiological down-regulation of AFP gene expression before birth. All these results show that this serum-free culture of rat yolk sac explants is a valuable system for further investigating the action of natural compounds and pharmacological drugs on endodermal gene expression during the embryonic and fetal periods.

Phorbol esters down-regulate alpha-fetoprotein gene expression without affecting growth in fetal hepatocytes in primary culture

The effects of phorbol esters (phorbol-12,13-dibutyrate, PDB) on alpha-fetoprotein expression and cell growth were assayed by using fetal hepatocytes in primary culture. PDB acts synergistically with epidermal growth factor (EGF) to specifically decrease alpha-fetoprotein (AFP) mRNA levels, without affecting the expression of other genes of the same family, such as albumin and Vitamin D-binding protein (DBP). This effect is PDB-dose dependent, maximal effects being at 10 ng/ml. The implication of protein kinase C (PKC) in this effect seems clear since bisindolylmaleimide (BIS), a specific PKC inhibitor, completely blocks the PDB effect on AFP expression. Nuclear run-on experiments show that the decrease in AFP mRNA levels is mainly due to an inhibition in the transcription rate of the gene. Determination of PKC activities shows that fetal hepatocytes contain mainly Ca 2+-independent isoenzymes, which patterns of activation was not modified by EGF plus PDB treatment with respect to PDB treatment. We have found that MAPK and JNK activities, c- jun and c- fos mRNA levels and AP-1 binding activity are notably increased when cells are incubated with both EGF and PDB. PDB does not stimulate growth of fetal hepatocytes, measured either as [3H]-thymidine incorporation into DNA or by cell cycle analysis using flow cytometry. All these results suggest that activation of PKC may affect liver gene expression rather than cell growth in fetal hepatocytes.

Quantitation of alpha-fetoprotein and albumin messenger RNA in human hepatocellular carcinoma

To analyze gene expression of alpha-fetoprotein (AFP) and albumin in hepatocellular carcinoma (HCC), messenger RNAs (mRNAs) of these proteins in six human hepatoma cell lines and in 30 cases of HCC were quantitatively analyzed by competitive reverse transcription (RT) followed by polymerase chain reaction (PCR). The transcriptional levels of both AFP and albumin genes in HepG2 and Huh 7 cell lines were 1010 copies/microgram RNA, in contrast to approximately 105 copies/microgram RNA in HLE and HLF cell lines. AFP and albumin mRNA levels in three normal livers were 105 and 1010 transcripts/microgram RNA, respectively. In 30 cases with HCC AFP mRNA level in neoplasm was 10 to 105-fold enhanced as compared with that of nonneoplastic portion, and correlated with serum AFP level and tumor size (P < .01). In contrast, albumin mRNA level was not reduced in the neoplasms presenting enhanced AFP mRNA levels, indicating that AFP and albumin gene expression in situ is not necessarily mutually exclusive. Prospective analysis revealed that an increased serum AFP was shown at the time of recurrence among patients with enhanced AFP mRNA levels in neoplasm only, indicating that AFP mRNA levels in neoplasm could be a clinically predictable tool. (Hepatology 1996 Jun;23(6):1384-92)

Fetal hepatic alpha-fetoprotein mRNA expression in fetuses with trisomy 21 and 18 at 12-15 weeks gestation.

In this study we examined alpha-fetoprotein (AFP) mRNA expression in fetal liver at 12-15 weeks of gestation in trisomy 21 (n = 13), trisomy 18 (n = 5) and control fetuses (n = 24). No significant difference was found in the steady-state level of fetal liver AFP mRNA levels in either of the two trisomy groups studied. These findings suggest that the decrease in maternal serum AFP concentration found in trisomic pregnancies is unlikely to be the consequence of impaired transcription of the AFP gene by the fetal liver.

各種成長因子によるＡＦＰ遺伝子発現の調節機構

Alpha-fetoprotein (AFP) and albumin gene expression are specific for hepatocytes. Since liver contains not only hepatocytes but also sinusoidal lining cells such endothelial cell, Kupffer cell, Ito cell and pit cell, the liver specific gene expression may be regulated by paracrine, autocrine and endocrine mechanism. In the present study, we analyzed the factors involved in the regulation of AFP and albumin gene expression using cultured human hepatoma cell lines. Epidermal growth factor and phorbol ester synergistically suppressed the AFP enhancer activity, resulting in the down-regulation of both AFP and albumin gene expression. Hepatocyte growth factor and transforming growth factor β1 suppressed the AFP gene expression through the inhibition of its promoter activity. Δ12-Prostaglandin J2, an active form of prostaglandin D2, repressed both AFP and albumin promoter activity. AFP and albumin mRNA levels were reversibly suppressed by the increased colloid osmotic pressure. Butyric acid, a natural fermentation product of colonic bacterial flora, inhibited the proliferation of HuH-7 human hepatoma cells in conjunction with the reciprocal regulation of AFP and albumin gene expression. These results indicate that AFP gene expression as well as albumin gene expression is physiologically regulated by varing factors in a paracrine fashion or an endocrine fashion through the arterial and the portal blood flow.