Gallbladder carcinoma with a large monolocular cystic cancerous component.

The monolocular cystic formation associated with gallbladder carcinoma is an extremely rare condition. A 79-year-old female suffering from upper abdominal pain and distention was admitted to our hospital. Ultrasonography and computed tomography revealed a monolocular cyst with an irregular wall thickness of 15 cm in diameter concomitant with a solid mass of 8 cm in diameter around the gallbladder bed. During celiotomy, the tumor was found to have a large pale gray cystic component at the fundus of the gallbladder, and disseminated nodules were observed in the peritoneum. We diagnosed the patient with gallbladder carcinoma and performed a simple cholecystectomy that included the tumor without systematic lymphadenectomy. On the cut face of the gallbladder, the lumen was occupied by a solid neoplasm. The cyst included a large amount of serous fluid and protruded continuously from the body of the gallbladder, but it did not communicate with the gallbladder lumen. Although the mechanism responsible for the development of cyst-forming papillary carcinoma of the gallbladder remains unknown, the present case is crucial for understanding the mechanism of cystogenesis in gallbladder carcinoma.

Signet ring cell carcinoma of the lower bile duct with rapid growth: report of a case.

Signet ring cell carcinoma occurring in the biliary tract is extremely rare. We herein report the case of a 78-year-old Japanese woman demonstrating signet ring cell carcinoma of the lower bile duct with a rapid growth. Computed tomography of the pancreas head pointed out a circular thickness in the lower bile ductal wall and stenosis of the common bile duct. Cholangiography revealed tapering stenosis at the lower bile duct. Biopsy specimens taken from these lesions and scratched specimens taken from stenotic portion of the lower bile duct were analyzed and demonstrated signet ring cell carcinoma. To the best of our knowledge, this is the first reported case of primary signet ring cell carcinoma of the lower bile duct reported in the English literature. Based on our experience, signet ring cell carcinoma of the lower bile duct is considered to demonstrate both transmural dispersion and an aggressive nature.

Recognition of nucleotide analogs containing the 7,8-dihydro-8-oxo structure by the human MTH1 protein.

The MTH1 protein catalyzes hydrolysis of oxidatively damaged purine nucleotides including 8-hydroxy-dGTP to the monophosphates. The MTH1 protein seems to act as an important defense system against mutagenesis, carcinogenesis, and cell death induced by oxidized purine nucleotides. We previously reported that the functional groups at the 2- and 6-positions of the purine ring affect the recognition by the human MTH1 protein. 8-Hydroxy-dGTP and 8-hydroxy-dATP are substrates of MTH1, and both have the "7,8-dihydro-8-oxo structure." In this study, three nucleotide analogs containing this motif were examined. A synthetic purine analog containing the 7,8-dihydro-8-oxo structure and the 2-amino function (dJTP) was hydrolyzed to the monophosphate with high efficiency by MTH1. On the other hand, two analogs that lack the two-ring system of their bases [formamidopyrimidine-dGTP (FAPY-dGTP) and 2-OH-dYTP] were poor substrates. FAPY-dGTP is a mixture of conformers and was hydrolyzed more than ten-fold less efficiently than 8-hydroxy-dGTP. These results clarify the effects of the 2-amino group and the two-ring system of the purine base on the recognition by the human MTH1 protein.

Atm heterozygosity cooperates with loss of Brca1 to increase the severity of mammary gland cancer and reduce ductal branching.

The role of homozygous ataxia telangiectasia mutated (ATM) mutations in familial and sporadic forms of cancer is well established, but the contribution of ATM heterozygosity to mammary gland and other cancers has been controversial. To test the effect of Atm heterozygosity on mammary gland cancer, mice with complete loss of exon 11 of Brca1 specifically in mammary epithelium (Brca1-MG-Deltaex11) were studied in either Atm heterozygous or Atm wild-type backgrounds. Targeted deletion of Brca1 in mammary epithelium resulted in carcinomas and adenocarcinomas of varying histology with long (>9 months) latency. Latency to tumorigenesis was found to be unchanged in the Brca1-MG-Deltaex11;Atm heterozygous mice compared with Brca1-MG-Deltaex11;Atm wild-type mice. However, the mice displayed variable tumor severity and differences in mammary tissue development. Mammary tumors from Brca1-MG-Deltaex11;Atm heterozygous mice were anaplastic and undifferentiated in all 20 tumors tested, whereas tumors from mice that were Brca1-MG-Deltaex11 but wild-type for Atm displayed variable histologic profiles, with some anaplastic tumors and other differentiated and less invasive tumor types. Previously reported developmental defects for Brca1-deficient mice were also observed in our model with and without Atm heterozygosity, but Brca1-MG-Deltaex11;Atm heterozygous mice displayed decreased ductal branching during puberty, a phenotype that was not observed in Brca1-MG-Deltaex11;Atm wild-type mice. Our results provide evidence that Atm heterozygosity influences severity of mammary gland tumors in the Brca1-MG-Deltaex11 tumor-prone mouse and suggest that this mutation leads to a newly characterized developmental defect during glandular maturation.

[Combination chemotherapy with oral TS-1 and low-dose CPT-11 by hepatic arterial infusion for multiple hepatic metastases from colon cancer--a case report].

Combined chemotherapy consisting of oral TS-1 and low-dose CPT-11 by hepatic arterial infusion is suggested to be a new effective treatment for multiple liver metastases from colorectal cancer. A 53-year-old man was diagnosed with multiple hepatic metastases from advanced colon cancer (Stage IV). The patient underwent partial resection of the colon and catheter insertion into hepatic artery for arterial infusion in November 2003. He was treated with postoperative combination chemotherapy consisting of UFT and low-dose CPT-11. UFT was administered orally at 400 mg/body/day everyday and CPT-11 was injected at 40 mg/body/week for 6 weeks, followed by a 2 weeks rest interval as 1 cycle. In spite of the reduction of metastatic liver tumors after 2 cycles of the chemotherapy, a metastatic pleural tumor appeared. Therefore, we judged the effect of the chemotherapy to be a progressive disease and changed UFT in the regimen to TS-1. TS-1 was administered orally at 80 mg/body/day under a 2-weeks-on and 1-week-off regimen for 3 times. CPT-11 was injected at 40 mg/body/week for 6 weeks, followed by a 3 weeks rest interval as 1 cycle. A stable disease was maintained for 3 months. Outpatient care was possible because no severe events were observed. Tumors showed a reduction rate of 37.4% after the combination therapy. The patient survived for 285 days after the operation.

Cancer chemoprevention by the antioxidant tempol acts partially via the p53 tumor suppressor.

We previously demonstrated that the nitroxide antioxidant tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) increased latency to tumorigenesis and doubled (100%) the lifespan of Atm-deficient mice, a mouse model of ataxia telangiectasia, which displays accelerated oxidative damage and stress. Tempol treatment of cancer-prone p53-deficient mice resulted in a small but significant (25%) increase in lifespan by prolonging latency to tumorigenesis, demonstrating that existing oxidative stress and damage are not necessary for the chemopreventative effects of tempol. However, the relatively small effect on latency in p53-deficient mice and the finding that tempol-mediated resistance to oxidative insult was p53-dependent suggested a more direct role of p53 in the chemopreventative effects of tempol. Surprisingly, tempol treatment specifically increased serine 18 phosphorylation of p53 (but not gamma-H2AX) and p21 expression in primary thymocytes in vitro in a p53-dependent fashion. Inhibition of phosphoinositide 3-kinase (PI3K) family members suggested that SMG-1 was responsible for the tempol-mediated enhancement of p53 serine 18 phosphorylation. These data suggest that the chemopreventative effect of tempol is not solely due to the reduction of oxidative stress and damage but may also be related to redox-mediated signaling functions that include p53 pathway activation.

Cancer chemoprevention by the antioxidant tempol in Atm-deficient mice.

Reactive oxygen species (ROS) are important endogenous etiological agents for DNA damage, and ROS perform critical signaling functions in apoptosis, stress responses and proliferation. The correlation between a lower incidence of cancer in people who consume a diet high in naturally occurring antioxidants and the observed increased ROS in cancerous tissues suggest that antioxidants may be used in cancer chemoprevention. We tested this hypothesis by determining whether the well-described nitroxide antioxidant, tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), acts as a chemopreventative agent in Atm mutant mice, a model of the human cancer prone syndrome ataxia-telangiectasia. Tempol administered continuously via the diet after weaning resulted in an increased lifespan of these mice by prolonging the latency to thymic lymphomas. Tempol treatment reduced ROS, restored mitochondrial membrane potential, reduced tissue oxidative damage and oxidative stress, consistent with antioxidant effects. In addition, this nitroxide lowered weight gain of tumor prone mice without changes in food intake, metabolism or activity level and exhibited an anti-proliferative effect in vitro. Thus, tempol acts as a novel chemopreventative agent in this mouse model of a human cancer prone syndrome, associated with broad antioxidant effects.

Probing the substrate recognition mechanism of the human MTH1 protein by nucleotide analogs.

To examine the substrate recognition mechanism of the human MTH1 protein, which hydrolyzes 2-hydroxy-dATP, 8-hydroxy-dATP, and 8-hydroxy-dGTP, ten nucleotide analogs (8-bromo-dATP, 8-bromo-dGTP, deoxyisoinosine triphosphate, 8-hydroxy-dITP, 2-aminopurine-deoxyriboside triphosphate, 2-amino-dATP, deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, and dUTP) were incubated with the MTH1 protein. Of these, the former five nucleotides were hydrolyzed with various efficiencies. The fact that the syn-oriented brominated nucleotides were hydrolyzed suggests that the MTH1 protein binds to deoxynucleotides adopting the syn-conformation. However, 8-hydroxy-dITP, which lacks the 2-amino group of 8-hydroxy-dGTP, was degraded with tenfold less efficiency as compared with 8-hydroxy-dGTP. In addition, deoxyisoinosine triphosphate, lacking the 6-amino group of 2-hydroxy-dATP, was hydrolyzed as efficiently as 8-hydroxy-dGTP, but less efficiently than 2-hydroxy-dATP. These results clarify the effects of the anti/syn conformation and the functional groups on the 2 and 6 positions of the purine ring on the recognition by the human MTH1 protein.

Combined loss of expression of O6-methylguanine-DNA methyltransferase and hMLH1 accelerates progression of hepatocellular carcinoma.

BACKGROUND AND OBJECTIVES: O(6)-methylguanine-DNA methyltransferase (MGMT) and human Mut L homologue 1 (hMLH1) are proteins that play an important role in DNA repair. No reports have yet described whether deficient MGMT and hMLH1 expression correlates with tumor progression and the prognosis of patients with hepatocellular carcinoma (HCC). METHODS: Using immunohistochemical analysis, we evaluated the expression status of MGMT and hMLH1 protein in 60 paraffin-embedded samples from consecutive patients with curatively resected HCC. RESULTS: The lack of expression of both MGMT and hMLH1 in HCCs (n = 7) correlated with advanced pTNM stage (P = 0.039), as compared with HCCs expressing both proteins (n = 25). The absence of both MGMT and hMLH1 was a significant indicator of malignant potential. The expression status of both MGMT and hMLH1 was a predictive factor for overall survival in patients with HCC (P < 0.001). CONCLUSIONS: HCC lacking both MGMT and hMLH1 is correlated with an advanced stage and a poor prognosis. The expression status of both repair proteins is a predictive prognostic marker in patients with HCC after surgical resection.

Role of tryptophan residues in the recognition of mutagenic oxidized nucleotides by human antimutator MTH1 protein.

The human MTH1 antimutator protein hydrolyzes mutagenic oxidized nucleotides, and thus prevents their incorporation into DNA and any subsequent mutation. We have examined its great selectivity for oxidized nucleotides by analyzing the structure of the protein and its interaction with nucleotides, as reflected in the fluorescence of its tryptophan residues. The binding of nucleotides decreased the intensity of MTH1 protein fluorescence and red-shifted the emission peak, indicating that at least one tryptophan residue is close to the binding site. Oxidized nucleotides (2-OH-dATP and 8-oxo-dGTP) produced a larger decrease in fluorescence intensity than did unoxidized nucleotides, and MTH1 protein had a much higher binding affinity for oxidized nucleotides. Deconvolution of protein fluorescence by comparison of its quenching by positively (Cs(+)) and negatively (I(-)) charged ions indicated that the MTH1 tryptophan residues are in two different environments. One class of tryptophan residues is exposed to solvent but in a negatively charged environment; the other class is partially buried. While the binding of unoxidized nucleotides quenches the fluorescence of only class 1 tryptophan residue(s), the binding of oxidized nucleotides quenched that of class 2 tryptophan residue(s) as well. This suggests that selectivity is due to additional contact between the protein and the oxidized nucleotide. Mutation analysis indicated that the tryptophan residue at position 117, which is in a negative environment, is in contact with nucleotides. The negatively charged residues in the binding site probably correlate with the finding that nucleotide binding requires metal ions and depends upon their nature. Positively charged metal ions probably act by neutralizing the negatively charged nucleotide phosphate groups. (c) 2002 Elsevier Science Ltd.

Deficient expression of O(6)-methylguanine-DNA methyltransferase combined with mismatch-repair proteins hMLH1 and hMSH2 is related to poor prognosis in human biliary tract carcinoma.

BACKGROUND: O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that transfers methyl groups from O(6)-methylguanine to itself. Alkylation of DNA at the O(6) position of guanine is an important step in the induction of mutations in the organism by alkylating agents. The O(6)-methyl G:T mismatch is recognized by the mismatch-repair (MMR) pathway. The biliary duct is highly exposed to alkylating agents because of its anatomical location. METHODS: We examined 39 surgically resected gallbladder carcinomas and 35 extrahepatic bile duct carcinomas and evaluated the expression of MGMT and MMR protein (hMLH1 and hMSH2) by immunohistochemical staining. RESULTS: MGMT-negative staining was detected in 59.0% of gallbladder carcinoma specimens and 60.0% of extrahepatic bile duct carcinoma specimens. In gallbladder carcinoma, hMLH1- and hMSH2-negative staining was observed in 51.3% and 59.0%, respectively, whereas in extrahepatic bile duct carcinoma, the respective values were 57.1% and 65.7%. MGMT-negative staining correlated with hepatic invasion in gallbladder carcinoma and with poor prognosis in both types of tumor. Furthermore, a combined MGMT and MMR status was shown to be a more significant prognostic biomarker in both tumor types. CONCLUSIONS: Combined MGMT and MMR is a possible prognostic marker that probably reflects an accumulation of genetic mutations.

8-Chloro-dGTP, a hypochlorous acid-modified nucleotide, is hydrolyzed by hMTH1, the human MutT homolog.

The human mutT homolog, hMTH1, suppresses spontaneous mutations by degrading the endogeneous mutagen, 8-hydroxy-dGTP. We previously reported the broad substrate specificity of hMTH1, which also degrades the oxidatively damaged purine nucleotides, 2-hydroxy-dATP, 8-hydroxy-dATP, 2-hydroxy-ATP, and 8-hydroxy-GTP, in addition to 8-hydroxy-dGTP. In this paper, we describe the hMTH1 activity for 8-chloro-dGTP, which could be formed in inflamed tissue by the reaction of dGTP with hypochlorous acid, a product of myeloperoxidase from activated human neutrophils. The hMTH1 protein was mixed with 1-20 microM of 8-chloro-dGTP and 8-hydroxy-dGTP, and the reaction products were quantified by anion-exchange HPLC to measure the pyrophosphatase reaction rate. The kinetic parameters revealed that 8-chloro-dGTP was degraded by hMTH1 with 50% efficiency as compared with that of 8-hydroxy-dGTP. This result suggests that 8-chloro-dGTP is an intrinsic substrate for hMTH1.

Substrate recognition by the human MTH1 protein.

A nucleotide pool sanitizing enzyme, the human MTH1 protein, hydrolyzes 2-hydroxy-dATP, 8-hydroxy-dATP, and 8-hydroxyd-GTP. To examine the substrate recognition mechanism of the MTH1 protein, ten nucleotide analogs (8-bromo-dATP, 8-bromod-GTP, deoxyisoinosine triphosphate, 8-hydroxy-dITP, 2-aminopurine-deoxyriboside triphosphate, 2-amino-dATP, deoxyxanthosine triphosphate, deoxyoxanosine triphosphate, dITP, and dUTP) were incubated with the protein. Of these, the former five nucleotides were hydrolyzed with various efficiencies. This results suggests the importance of the anti/syn-conformation and the functional groups on the 2 and 6-positions of the purine ring.