Expression and bioactivity of human α-fetoprotein in a Bac-to-Bac system.

α-fetoprotein (AFP) is an early serum growth factor in foetal embryonic development and hepatic oncogenesis. A growing number of investigations of AFP as a tumour-specific biomarker have concluded that AFP is an important target for cancer treatment. AFP also plays an immunomodulatory role in the treatment of several autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis and thyroiditis. In an effort to support biochemical screening and drug design and discovery, we attempted to express and purify human AFP in a Bac-to-Bac system. Two key factors affecting the expression of recombinant human AFP (R-AFP), namely the infectious baculovirus inoculum volume and the culturing time post-infection, were optimized to maximize the yield. We achieved a high yield of approximately 1.5 mg/l of harvested medium with a 72-96 h incubation period after infection and an inoculum volume ratio of 1:100. We also assessed the role of R-AFP in the proliferation of the human liver cancer cell line Bel 7402, and the results indicated that R-AFP promoted the growth of hepatoma cells. We concluded that this method can produce high yields of R-AFP, which can be used for studies related to AFP.

Effects of sodium thiosulfate on vascular calcification in end-stage renal disease: a pilot study of feasibility, safety and efficacy.

BACKGROUND AND OBJECTIVES: Vascular calcification is a major contributor to morbidity and mortality in hemodialysis. The objective of this pilot study was to determine the feasibility, safety and efficacy of sodium thiosulfate (STS) in the progression of vascular calcification in hemodialysis patients. METHODS: Chronic hemodialysis patients underwent a battery of cardiovascular tests. Those with coronary artery calcium (Agatston scores >50) received intravenous STS after each dialysis for 5 months (n = 22) and the tests were repeated. Changes in MDCT-determined calcification were assessed as the mean annualized rate of change in 3 vascular beds (coronary, thoracic and carotid arteries) and in L1-L2 vertebral bone density. RESULTS: Although individual analyses showed coronary artery calcification progression in 14/22 subjects, there was no progression in the mean annualized rate of change of vascular calcification in the entire group. The L1-L2 vertebral bone density showed no changes. There were no correlations between rates of progression of vascular calcification and phosphorus, fetuin or C-reactive protein levels. Changes in coronary artery calcification scores correlated with those of the thoracic aorta. CONCLUSION: STS treatment is feasible, appears safe and may decrease the rate of progression of vascular calcification in hemodialysis patients. A large, randomized, controlled trial is warranted.

[Complete remission with FLEP chemotherapy for multiple liver metastasis from alpha-fetoprotein-producing gastric cancer--report of a case].

The patient was a 51-year-old male diagnosed with gastric cancer in July 1999 by endoscopic examination, revealing multiple liver metastasis with abdominal computed tomography (CT). The serum levels of alpha-fetoprotein (AFP)were determined to be 91 ng/mL, and tumors were histopathologically identified as AFP-producing gastric cancer by immunohistological staining. We started combination chemotherapy with 5-fluorouracil (5-FU), Leucovorin (LV), etoposide (VP-16) and cis-diaminedichloroplatinum (CDDP) (designated as FLEP)in August 1999. The serum AFP value was normalized after two courses, and the liver metastases disappeared. The primary gastric tumor became a ulcer, and disappearance of the cancer was confirmed histologically. We continued adjuvant chemotherapy with S-1 as an outpatient. In April 2000, there was no sign of the liver metastases, but endoscopic examination showed IIc-like lesion in the stomach. We performed 2 courses of FLEP, but the tumor did not disappear. He underwent total gastrectomy with D2 dissection in June 2001. The pathological diagnosis was por 1, ss, ly2, v1, n(1+). He was still alive with no sign of recurrence 84 months after surgery. We experienced this AFP-producing gastric cancer in which CR was possible by FLEP. There was no recurrence after total gastrectomy for local recurrence.

Processing and transport of matrix gamma-carboxyglutamic acid protein and bone morphogenetic protein-2 in cultured human vascular smooth muscle cells: evidence for an uptake mechanism for serum fetuin.

Matrix gamma-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent gamma-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Human vascular smooth muscle cells (VSMCs) were infected with an adenovirus carrying the MGP construct, which produced non-gamma-carboxylated MGP and fully gamma-carboxylated MGP. Both forms of MGP were found in the cytosolic and microsomal fractions obtained from the cells by differential centrifugation. The crude microsomal fraction was shown to contain an additional, more acidic Ser-phosphorylated form of MGP believed to be the product of Golgi casein kinase. The data suggest that phosphorylation of MGP dictates different transport routes for MGP in VSMCs. A proteomic approach failed to identify a larger soluble precursor of MGP or an intracellular carrier protein for MGP. Evidence is presented for a receptor-mediated uptake mechanism for fetuin by cultured human VSMCs. Fetuin, shown by mass spectrometry not to contain MGP, was found to be recognized by anti-MGP antibodies. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.

[Hepatic metastases from alpha-fetoprotein producing colorectal cancer that responded remarkably to transcatheter arterial chemo-embolization].

Although alpha-fetoprotein (AFP)-producing colorectal cancer is extremely rare, its potential for liver metastasis is high and the prognosis of the patient is very poor. We report a case of metastatic liver tumor from an AFP producing sigmoid colon cancer that was successfully treated with transcatheter arterial chemo-embolization (TACE). A 48-year-old female was admitted to our hospital complaining of anal bleeding, and was diagnosed as harboring a type 2 advanced tumor with two metastatic lesions in the lateral segment of the liver. Serum AFP measured 948.2 ng/ml before surgery. We performed sigmoidectomy and lateral segmentectomy of the liver. Pathological examination revealed moderately differentiated adenocarcinoma both in the primary and the metastatic lesions, and AFP-stain positive nests were seen on immunohistochemical staining. The serum AFP value returned to normal soon after the surgery. Five months later, a recurrent lesion was discovered in the right lobe of liver, and it was surgically removed. Another 4 months later, multiple liver metastases were recognized and the patient's serum AFP level was found to be 593.3 ng/ml. For the re-recurrent hepatic lesions of 5.5 cm in diameter, she underwent two courses of TACE using Lipiodol and epirubicin hydrochloride. Following the treatment, the tumor shrank rapidly and almost disappeared. The patient has been in good health for 24 months since the last TACE, and the serum AFP level has been within the normal range.

Fetuin-B, a second member of the fetuin family in mammals.

A set of orthologous plasma proteins found in human, sheep, pig, cow and rodents, now collectively designated fetuin-A, constitutes the fetuin family. Fetuin-A has been identified as a major protein during fetal life and is also involved in important functions such as inhibition of the insulin receptor tyrosine kinase activity, protease inhibitory activities and development-associated regulation of calcium metabolism and osteogenesis. Furthermore, fetuin-A is a key partner in the recovery phase of an acute inflammatory response. We now describe a second protein of the fetuin family, called fetuin-B, which is found at least in human and rodents. On grounds of domain homology, overall conservation of cysteine residues and chromosomal assignments of the corresponding genes in these species, fetuin-B is unambiguously a paralogue of fetuin-A. Yet, fetuin-A and fetuin-B exhibit significant differences at the amino acid sequence level, notably including variations with respect to the archetypal fetuin-specific signature. Differences and similarities in terms of gene regulation were also observed. Indeed, studies performed during development in rat and mouse showed for the first time high expression of a member of the fetuin family in adulthood, as shown with the fetuin-B mRNA in rat. However, like its fetuin-A counterpart, the fetuin-B mRNA level is down-regulated during the acute phase of experimentally induced inflammation in rat.

[A case of AFP-producing gastric cancer after curative operation effectively treated with chemotherapies including hepatic arterial infusion therapy].

A 63-year-old woman was admitted complaining of epigastralgia. An endoscopic examination revealed a Borrmann type 2 lesion at the greater curvature of the middle third in the stomach. Abdominal computed tomography detected no liver metastasis. The preoperative serum alpha-phetoprotein (AFP) level was elevated to 242.9 ng/ml. 5-fluorouracil (5-FU) was given 200 mg/day for 26 days orally. Distal gastrectomy with D3+ No. 16b1 lymph node dissection, cholecystectomy and hepatic arterial canulation were performed, and mitomycin C 20 mg was injected intravenously during the operation. Immunohistochemical staining of the specimen for AFP by the SAB method was positive in the cancer lesion. After the operation, FP (cisplatin 100 mg on day 1, 5-FU 750 mg on days 1-3) therapy of one course and intrahepatic arterial infusion therapy using adriamycin 10-20 mg every 2 weeks for 7 months were conducted. Moreover, the patient took UFT 400 mg/day for 26 months orally on an outpatient basis as an adjuvant chemotherapy. The only toxicity was neutropenia (grade 3), but it abated without an interruption in the chemotherapy. The AFP level declined gradually, and returned to the normal range 1 month after surgery. The patient is still alive with no sign of hepatic metastasis or recurrence 7 years and 7 months after the gastrectomy.

Regulation of alpha-foetoprotein gene expression by fatty acids and fibrates.

Alpha-foetoprotein (AFP), the major plasma protein in the foetus, is mainly synthesized by yolk sac and foetal liver. It binds polyunsaturated fatty acids and probably controls their metabolism and action. We investigated the effects of fatty acids and fibrates on expression of the AFP gene using two complementary approaches. Treatment with 5-8-11-14 eicosatetraynoic acid (ETYA), an analogue of arachidonic acid, specifically led to lower AFP mRNA levels in cultured rat yolk sac explants whereas treatment with palmitic or oleic acid did not. Clofibric acid and fenofibrate also gave lower AFP mRNA levels. Transient transfection experiments with HepG2 hepatoma cells showed that ETYA and clofibric acid decreased the transcriptional activity of the 7 kb regulatory region of the rat AFP gene. The 330 bp AFP promoter was identified as a target for these down regulating effects.

Differential expression of insulin receptor tyrosine kinase inhibitor (fetuin) gene in a model of diet-induced obesity.

The Differential Display technique has been used to identify differences in mRNA expression in adipose tissue after the introduction of a high fat diet to two strains of rat (OM and S5B/PI) that differ in their susceptibility to develop obesity on this diet. The insulin receptor tyrosine kinase inhibitor protein (fetuin) was shown to be differentially expressed in OM but not S5B/PI rats. This circulating protein may play a role in the development of peripheral insulin resistance associated with high fat diets.

Purification and characterization of human and mouse recombinant alpha-fetoproteins expressed in Escherichia coli.

Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function(s) remains unclear. A more complete analysis of the physiological activities of this oncofetal protein has, until now, been severely limited by the lack of an appropriate source from which to obtain pure AFP in any sizeable quantity. In the present investigation, we obviate this problem by cloning and efficiently overexpressing mature mouse and human AFP cDNA's in Escherichia coli. For recombinant mouse AFP (rMoAFP), large segments of the coding region were excised from the preexisting plasmids pAFP1 and pAFP2, which together encompass 90% of the AFP sequence. The mouse cDNA was made complete by the addition of N- and C-terminal encoding oligonucleotides. Mouse AFP cDNA was expressed directly as a full-length molecule in vector pTrp4 or as fusion proteins in plasmids pMALc and pRX1 under the transcriptional control of trp or tac promoters. Accumulation of rMoAFP was significantly increased in protease-deficient E. coli strains over nonprotease-deficient strains, > or = 10% of total cell protein. Of the gene fusion proteins examined, none offered significant advantage over the direct expression product in terms of recombinant protein stability, overall levels of synthesis, or facilitated purification. Recombinant AFP polypeptides expressed by pTrp4 were as expected, deposited in bacterial inclusion bodies. Subsequent to resolubilization/refolding, rMoAFP was first enriched by passage over Q-Sepharose resin followed by final purification using immobilized copper-chelate affinity chromatography. Protein sequencing of the N-terminus revealed that purified rMoAFP had a deletion of the first nine amino acids coded for by the full-length mouse AFP cDNA. Similar N-terminal deletions are observed with AFP isolates originating from natural sources. A complete human AFP cDNA was generated from a fetal liver cDNA library and was cloned into vector pTrp4. Recombinant human AFP (rHuAFP) was expressed under the identical conditions employed for rMoAFP but purification had to be modified to include preparative Mono Q anion exchange chromatography. N-terminal sequencing, amino acid compositional analysis, and electrospray mass spectrometry revealed that purified rHuAFP was intact and unaltered and that the initiator methionine was completely removed. The biological activity of recombinant AFP, as judged by its inhibitory effects on in vitro lymphocyte proliferation, was equivalent to that of the native protein. The availability of large quantities of mouse and human recombinant AFP molecules should now permit detailed structure-function analyses of this important oncofetal protein to proceed in a manner unimpeded by previous limitations in both quantity and quality of the native proteins.

[Effective transcatheter arterial embolization for hepatic metastasis in a case of AFP producing gastric cancer].

We experienced a case of effective transcatheter arterial embolization (TAE) for hepatic metastasis in a AFP producing gastric cancer. A 51-year-old man with 2 type gastric cancer (H0) underwent subtotal gastrectomy (D2). The serum AFP level was 134.3 ng/ml, and AFP positive tumor cells were detected by PAP method. After the operation, the serum AFP level initially decreased but re-increased on the 7th postoperative month, and metastatic lesions of the liver were detected by CT scan. After the patient was treated 4 times by TAE, the serum AFP level returned within normal range and the metastatic tumors of the liver decreased markedly. Therefore liver resection was performed at the 28th month after the first operation. Total necrosis of metastatic liver lesions was confirmed. This patient has been well without recurrence signs for 10 years since operation. It is concluded that TAE should be used to treat hepatic metastasis in the case of an AFP producing gastric cancer.

Hepatic regeneration in vitamin A-deficient rats: changes in the expression of transforming growth factor alpha/epidermal growth factor receptor and retinoic acid receptors alpha and beta.

We have studied the effect of vitamin A deficiency on the expression of transforming growth factor alpha (TGF-alpha), hepatocyte growth factor, acidic fibroblast growth factor, and TGF-beta 1 after partial hepatectomy of vitamin A-supplemented and vitamin A-deficient rats. In addition, the expressions of epidermal growth factor receptor and retinoic acid receptors alpha (RAR alpha) and beta (RAR beta) were studied. Partial hepatectomy was performed on the animals from the vitamin A-supplemented and -deficient groups at the age of 10 weeks when the weights of the animals on the deficient diet had reached a plateau. Two animals from each group were sacrificed before the operation and also 12, 24, 48, and 72 h and 5 days after the operation. Partial hepatectomy of the vitamin A-deficient rats leads to a focal necrosis of liver followed by a rapid restoration of liver mass. Expression of the TGF-alpha and epidermal growth factor receptor was highly elevated in the livers of deficient animals after partial hepatectomy. In the vitamin A-supplemented animals, the level of epidermal growth factor receptor was down-regulated following partial hepatectomy. Proliferation of oval cells in vitamin A-deficient livers following partial hepatectomy and subsequent increase in 2.1-kilobase alpha-fetoprotein mRNA was observed, suggesting an activation of the stem cell compartment. Another unexpected result was an inverse relationship between RAR beta and RAR alpha expression, the latter becoming the major species after partial hepatectomy in animals on the vitamin A-deficient regimen.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of interleukin 6 from human liver cell lines: production of interleukin 6 is not concurrent with the production of alpha-fetoprotein.

The production of interleukin (IL) 6 from six human liver cell lines, including Chang liver, HLF, HLE, HepG2, PLC/PRF/5, and HuH-7, was investigated using enzyme-linked immunosorbent assay and Northern blot analysis. When cells were cultured in the presence of 12-O-tetradecanoylphorbol-13-acetate, significant amounts of IL6 were detected in the culture supernatants of Chang liver cells, HLF cells, and HLE cells. However, IL6 was not detected in the culture supernatants from HepG2 cells, PLC/PRF/5 cells, or HuH-7 cells which had been treated similarly. To further investigate the production of IL6, expression of the IL6 gene was studied. Results of Northern blot analysis using IL6 complementary DNA as a probe showed that the induction was initiated at the mRNA level. Moreover, IL6 mRNA was also induced by IL1 beta and tumor necrosis factor but not by a calcium ionophore (A23187) or IL6 itself in Chang liver cells. This is the first study to demonstrate the production of human IL6 in liver cells. Furthermore, when the production of alpha-fetoprotein (AFP) from the liver cell lines was examined, the three that were able to produce IL6 failed to produce AFP, whereas the other three cell lines succeeded in producing AFP. These observations may indicate the heterogeneous origin of the liver cell lines.

Zhi-mu saponin inhibits alpha-fetoprotein gene expression in developing rat liver.

1. A saponin isolated from the Chinese herb zhi-mu (Anemarrhena asphodeloides Bunge) modifies alpha-fetoprotein production when injected into newborn rats. 2. The serum level of AFP was determined quantitatively by immunorocket electrophoresis. 3. AFP serum levels were reduced to 60% of the control by zhi-mu saponin (ZMS). 4. The lower AFP level in drug treated rat serum is not due to a change in the pattern of serum AFP variants. 5. AFP mRNA levels in ZMS-treated rat livers, measured by RNA dot hybridization, decreased to about 50% of control levels after 4 days treatment. 6. Results from tritium labeled dexamethasone competition assays suggest that ZMS may act on AFP gene expression through glucocorticoid receptor mediated action.

[Effects of various factors on the growth and function of a human hepatoblastoma cell line, HuH-6--an approach for serum-free cell culture].

The effects on a human hepatoblastoma cell line (HuH-6) of serum and Ca2+ were investigated. A higher concentration of serum reduced the production of alpha-fetoprotein, whereas Ca2+ enhanced that of both albumin and alpha-fetoprotein. In view of these facts, a serum-free medium using RPMI-1640 supplemented with lactalbumin hydrolysate, epidermal growth factor, hydrocortisone, insulin, chrelatoxine, glucagone and selenium was then developed. Collagen type IV was superior to collagen type I, laminin, fibronectin and plastic in supporting the growth of HuH-6 in the serum-free medium. Higher amounts of both albumin and alpha-fetoprotein were secreted in HuH-6 cultured in serum-free medium than in serum-supplemented medium.

Rapid development of large numbers of alpha-fetoprotein-containing "oval" cells in the liver of rats fed N-2-fluorenylacetamide in a choline-devoid diet.

Fischer rats, fed 0.05% w/w N-2-fluorenylacetamide in a choline-devoid diet for 2 weeks, develop a massive infiltration of the liver by small "oval" cells. This occurs rapidly one week after feeding the diet for two weeks. All rats fed choline-devoid diet die within 5 weeks, with massive oval cell infiltration of the liver. Although similar changes occur in rats fed N-2 fluorenylacetamide in a choline-supplemented diet, their degree is much less. In rats fed a choline-devoid diet without N-2-fluorenylacetamide, proliferation of hepatocytes, but not of oval cells, is observed. Because the carcinogen-enhancing effects of choline-devoid diets seem to exceed those of partial hepatectomy, such diets may work by causing changes distinct from those induced by partial hepatectomy. Many oval cells contain alpha-fetoprotein, and the rapid oval cell increase is associated with an exponential increase in serum alpha-fetoprotein concentration. These observations suggest that a cellular change, not an alteration of gene expression in parenchymal cells, is the primary cause of hyper-alphafetoproteinemia during the course of chemical carcinogenesis in rats.