Delta15N of soil N and plants in a N-saturated, subtropical forest of southern China.

We investigated the delta(15)N profile of N (extractable NH(4)(+), NO(3)(-), and organic N (EON)) in the soil of a N-saturated subtropical forest. The order of delta(15)N in the soil was EON > NH(4)(+) > NO(3)(-). Although the delta(15)N of EON had been expected to be similar to that of bulk soil N, it was higher than that of bulk soil N by 5 per thousand. The difference in delta(15)N between bulk soil N and EON (Delta(15)N(bulk-EON)) was correlated significantly with the soil C/N ratio. This correlation implies that carbon availability, which determines the balance between N assimilation and dissimilation of soil microbes, is responsible for the high delta(15)N of EON, as in the case of soil microbial biomass delta(15)N. A thorough delta(15)N survey of available N (NH(4)(+), NO(3)(-), and EON) in the soil profiles from the organic layer to 100 cm depth revealed that the delta(15)N of the available N forms did not fully overlap with the delta(15)N of plants. This mismatch in delta(15)N between that of available N and that of plants reflects apparent isotopic fractionation during N uptake by plants, emphasizing the high N availability in this N-saturated forest.

Allelic loss at 19q12 and Xq11-12 predict an adverse clinical outcome in patients with mucinous ovarian tumours of low malignant potential.

Ovarian tumours of low malignant potential (LMP) are intermediate between adenomas and ovarian carcinomas. These tumours are often associated with a significantly better prognosis than ovarian carcinomas. However, a subset of these tumours can progress and become lethal. In order to seek sensitive diagnostic tools for monitoring patients after surgical operation, we performed a genome-wide scan for loss of heterozygosity (LOH) in 41 mucinous LMPs using 91 polymorphic microsatellite markers at an average interval of 50 cM across all of the human chromosomes and 25 LOH markers reportedly associated with ovarian carcinoma. In addition, we assessed whether clinicopathological parameters, microvessel density, Ki-67 labeling index, apoptotic index or p53 overexpression would be useful for predicting the postoperative outcome of LMP patients. Of the 116 markers examined, 19q12 and Xq11-12 showed significant correlation between postoperative progression-free survival time and LOH status (P<0.05). Patients with a high Ki-67 labeling index had a significantly poorer progression-free survival time than those with lower levels (P=0.042). Other clinicopathological factors and immunohistochemical analysis had no correlation with progression-free survival time in this series of patients. When the combination of LOH at 19q12 and/or Xq11-12 was assessed using Cox's regression analysis, patients with tumours that showed LOH at these positions were at greatest risk of progression (P=0.0073). These findings suggest that the identification of LOH at 19q12 and/or Xq11-12 in former mucinous LMP sites should alert the clinician to the presence of a potentially aggressive lesion in the coelomic epithelium, even if a distinction between second primary tumours or recurrence could not be determined.

Comprehensive allelotype study of ovarian tumors of low malignant potential: potential differences in pathways between tumors with and without genetic predisposition to invasive carcinoma.

Ovarian tumors of low malignant potential (LMP) are intermediate between adenomas and ovarian carcinomas (OC); however, the relevance of LMP to ovarian carcinogenesis is not clear. We performed a comparative analysis of allelotypes in 50 cases of LMP (42 mucinous and 8 serous) and 23 cases of OC (15 mucinous and 8 serous) to investigate any differences in genetic changes. Analysis of loss of heterozygosity (LOH) using 25 microsatellite markers reportedly associated with OC revealed that the total LOH frequency at each marker was significantly lower in LMP than in OC (p < 0.01). However, 9 (36%) loci showed higher LOH frequency in mucinous LMP than in mucinous OC. A genome-wide scan for LOH using 91 microsatellite markers and fine mapping revealed that LOH at D7S1805 (7q35) is characteristic of mucinous LMP (19.4% in mucinous LMP, 8.3% in mucinous OC). We further studied LOH in 3 cases of mucinous OC that were accompanied by mucinous LMP lesions. In 2 cases, LOH frequency was higher in the carcinoma portion than in the morphologically LMP portion. The other case showed microsatellite instability in the morphologically LMP portion and LOH in the carcinoma portion. Our results suggest the presence of an LMP-to-OC developmental sequence and the existence of a subset of LMP that does not develop into OC in the mucinous subtype of ovarian tumors.

Loss of heterozygosity of nucleotide excision repair factors in sporadic ovarian, colon and lung carcinomas: implication for their roles of carcinogenesis in human solid tumors.

The deficiencies of nucleotide excision repair (NER) factors are genetic diseases, xeroderma pigmentosum (XP) increasing risk of developing cancer on sun-exposed areas of the skin. However, the abnormality of NER factors in human sporadic carcinoma remains unclear. Loss of heterozygosity (LOH) analysis for the XP, XPA, XPB, XPC, XPD, XPE, XPF, XPG and the transcription-coupled repair factor, Cockayne syndrome B (CSB) revealed that NER factors were abnormal in 62.1 % of ovarian tumors (18/29), 16.7% of colon (2/12) and 22.2% lung (2/9) carcinomas. Furthermore, 13.8% of ovarian, 8.3% of colon and 22% of lung carcinomas exhibited LOH for NER factors without LOH for tumor suppressor genes such as p53, FHIT, APC, BRCAI, BRCA2 and DCC. Although both microsatellite instability and LOH of NER factors were observed in some cases, there was no strong association between them in the present study. These observations raise the possibility that alterations of NER factors may be frequent in human sporadic carcinomas. Further study should be needed to find the direct evidence of NER gene abnormalities in human sporadic carcinoma tissues.

Expression and cisplatin sensitivity of copper-transporting P-type adenosine triphosphatase (ATP7B) in human solid carcinoma cell lines.

One of the most important clinical problems in the treatment of human solid carcinoma is the intrinsic/acquired resistance. Cisplatin is a platinum compound that is one of the most effective agents in clinic. Copper-transporting P-type adenosine triphosphate (ATP7B) has been reported to be associated with cisplatin resistance by the experiment of transfection of full cDNA of ATP7B into KB3-1 lacking ATP7B. We examined the relationship between mRNA expression level of ATP7B and sensitivity to cisplatin in nine human ovarian carcinoma cell lines to extend these findings. mRNA expression level of ATP7B was significantly correlated with cisplatin-sensitivity in nine cell lines, raising the possibility that ATP7B could be a chemoresistance marker in some types of human solid carcinoma.

Loss of heterozygosity of nucleotide excision repair factors in sporadic oral squamous cell carcinoma using microdissected tissue.

The deficiencies of nucleotide excision repair (NER) factors are involved in rare genetic diseases such as xeroderma pigmentosum (XP) with increased risk of developing cancer on sun-exposed areas of the skin. However, the abnormality of NER factors in human sporadic carcinoma remains unclear. Loss of heterozygosity (LOH) analysis, using the microdissected tissues, for the XPA, XPB, XPC, XPD, XPE, XPF, XPG and the transcription-coupled repair factor, Cockayne syndrome B (CSB) revealed that NER factors were abnormal in 30.0% (3/10 cases) of oral squamous cell carcinomas. Furthermore, 10.0% of oral carcinomas exhibited LOH for NER factors without LOH for tumor suppressor genes such as p53, FHIT, APC, BRCA1, BRCA2 and DCC. These observations raise the possibility that alterations of NER factors may be involved in carcinogenesis in human oral squamous cell carcinoma.

Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair.

While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.

Different features of angiogenesis between ovarian and breast carcinoma.

Angiogenesis assessed by immunohistochemical staining for endothelial cells has been widely accepted as an independent prognostic factor in human breast carcinoma. However, the clinicopathologic significance of angiogenesis is still being argued in ovarian carcinoma. Therefore, we retrospectively analyzed the clinicopathologic significance of angiogenesis in ovarian carcinoma compared with that in breast carcinoma. After vessels were stained with CD34-monoclonal antibody, the areas with the highest number of intratumoral microvessels were assessed in a 200x field in 42 ovarian carcinoma and 41 breast carcinoma. Intratumoral microvessel density (IMD) in ovarian carcinoma was significantly lower than that in breast carcinoma. Further, the difference of IMD from tumor to tumor in ovarian carcinoma was smaller than that in breast carcinoma. IMD was correlated with tumor grade, but not with other clinicopathologic variables in ovarian carcinoma. Although the patients with high-IMD tumor revealed a poorer prognosis than those with low-IMD tumor in breast carcinoma, IMD had no influential effects on the survival of the patients with ovarian carcinoma. Our comparative analysis of IMD in ovarian carcinoma with that in breast carcinoma indicates that angiogenesis may play an important role in the transient of ovarian neoplasms, but not in the progression of ovarian carcinomas, and that the biological roles of angiogenesis might be different, depending on histologic subtype.

Effect of intratumoral OK-432 administration of thymidine phosphorylase expression in human gastric carcinoma.

It is known that thymidine phosphorylase (dThdPase) is increased in various types of malignant tumors and is induced by cytokines. In this study, we have investigated the effects of OK-432, which induces multiple cytokines, on dTHdPase expression and angiogenesis in human gastric carcinomas. We examined 25 patients who underwent gastrectomy for gastric carcinoma. OK-432 was directly injected in tumors in 16 (OK group) of 25 patients via endoscopy before operation and the other 9 patients were not treated (control group). The dThdPase activity in carcinoma tissues of the OK group was significantly higher than that of the control group (P < 0.05). The amounts of IL-1 alpha, IFN-alpha, and IFN-gamma in carcinomas in the OK group were significantly higher than in the controls (P < 0.05), and these were significantly correlated with the dThdPase activity. Intratumoral OK-432 administration enhances the expression of dThdPase in gastric carcinoma cells by inducing various cytokines.

Expression of multidrug resistance-related transporters in human breast carcinoma.

The expression levels of mRNA for multidrug resistance 1 (MDR1) gene, multidrug resistance protein 1 (MRP1), lung resistance-related protein (LRP) and breast cancer resistance protein (BCRP), which confer multidrug resistance in vitro, were examined in 43 untreated breast carcinoma patients, of whom 38 subsequently received doxorubicin-based chemotherapy after surgery, in order to elucidate the roles of these genes in drug resistance in vivo. The mRNA levels were determined using a semi-quantitative reverse-transcription polymerase chain reaction method in breast carcinoma tissues including at least 80% carcinoma cells. The expression level of BCRP gene was low and did not vary markedly in comparison with that of MDR1, MRP1 or LRP gene. The expressions of MDR1 and MRP1 genes were correlated with each other, but the expression of BCRP or LRP gene did not correlate with that of other genes. These four gene expressions were independent of age, TNM categories and the status of progesterone or estrogen receptor. The expression levels of these four genes were not related to the relapse or prognosis of the 38 patients treated with doxorubicin-based chemotherapy. P-glycoprotein (P-gp) / MDR1, MRP1 and LRP may play more important roles than BCRP in chemotherapy of human breast carcinoma.

Preoperative assessment of vascularity by color Doppler ultrasonography in human rectal carcinoma.

PURPOSE: Although angiogenesis assessed by immunostaining endothelial cells (microvessel density) is a well-known prognostic factor in a wide variety of human solid tumors, preoperative determination of microvessel density seems to be difficult in rectal carcinoma. Thus, we performed transanal color Doppler ultrasonography in 46 patients with rectal carcinoma to assess preoperative angiogenic status and compare it with microvessel density in surgical specimens. METHODS: Time-averaged maximal velocity, peak systolic velocity, number of vascular points, and vascular point index were conducted by color Doppler ultrasonography in 46 patients with rectal carcinoma. Number of vascular points was defined as the number of vessels with pulsation in the section of tumor. Vascular point index was defined as the average number of vascular points divided by the area assessed by color Doppler ultrasonography in the section of tumor. The profiles of number of vascular points were similar to those assessed by microangiography in five rectal carcinomas. RESULTS: Vascular point index significantly correlated with microvessel density (P < 0.0001). No significant correlation was found between microvessel density and time-averaged maximal velocity or peak systolic velocity. Vascular point index was also a better indicator of lymph node metastasis and venous invasion than microvessel density. In addition, 11 of 46 cases with postoperative hematogenous metastasis (23.9 percent) were observed prospectively. Vascular point index may be a best predictor for hematogenous metastasis from rectal carcinoma compared with peak systolic velocity, time-averaged maximal velocity, and microvessel density by receiver operating characteristic analysis. CONCLUSION: These results suggest that preoperative quantification of angiogenesis using color Doppler ultrasonography will provide quick and useful information in the management of rectal carcinoma.

Allelotype analysis of gallbladder carcinoma associated with anomalous junction of pancreaticobiliary duct.

Anomalous junction of pancreaticobiliary duct (AJPBD) patients has an increased risk of gallbladder and bile duct carcinomas. However, the relevance of carcinoma with AJPBD is not fully clarified. We performed analysis of loss of heterozygosity (LOH) at p53 locus and immunohistochemistry of p53 and K-ras gene mutation in five cases of gallbladder carcinoma associated with AJPBD. LOH of p53 locus and overexpression of p53 were detected in two out of five (40%) and five out of five (100%), respectively, in the present study. K-ras gene mutation at codon 12 and 13 was not detected (0%, 0/5). These results suggest that aberrations of p53 are involved in carcinogenesis of gallbladder carcinoma associated with AJPBD. Next, in order to find the genetic events besides K-ras mutation and overexpression of mutant p53 in this disease, LOH analysis was performed using 72 microsatellite markers. High frequency of allelic loss (> 50%) was found on 2p (81.8%), 4p (50%), 4q (50%), 8q (60%), 9q (50%), 10p (50%), 14p (60%), 14q (50%), 16p (60%), 19p (50%), 21p (50%) and Xp (66.6%). The highest deletion regions on chromosome 2p24 (3/3, 100%), 14q22 (3/4, 75%) and 21q22 (3/4, 75%) were found. The present study suggests that gallbladder carcinoma associated with AJPBD has high frequent allelic loss and has two new regions which may harbor putative tumor suppressor genes.

Clinicopathological and prognostic significance of thymidine phosphorylase and proliferating cell nuclear antigen in gastric carcinoma.

We have reported that thymidine phosphorylase (dThdPase) is identical to platelet derived-endothelial cell growth factor and that it has an angiogenic activity in vitro and in human carcinoma tissues as well as gastric carcinoma. Recently, we revealed that dThdPase may have an another function(s) besides angiogenesis in vitro and in human solid tumors. Using immunohistochemistry, we examined retrospectively whether the expression of dThdPase was correlated with tumor growth, comparing it with the proliferating cell nuclear antigen labeling index (PCNA LI) and examining their prognostic significance in 116 patients with gastric carcinoma. A direct correlation of these two factors was observed (R=0.659, P<0.001). A multivariate Cox regression analysis showed that both dThdPase positivity and PCNA LI were independent prognostic factors, as were depth of invasion and lymph node metastasis. Furthermore, the patients with dThdPase-positive/high PCNA LI tumors had the worst prognoses. The combination of dThdPase and PCNA expression is a better tool for predicting the prognosis of patients with gastric carcinoma than the expression of either of them alone. These results raise the possibility that dThdPase may have a function(s) involved in tumor growth besides angiogenesis.

Characterization of a novel topoisomerase I mutation from a camptothecin-resistant human prostate cancer cell line.

In this study, we characterized the structure and function of topoisomerase I (top1) protein in the camptothecin (CPT)-resistant prostate cancer cell lines, DU-145/RC0.1 and DU-145/RC1 (RC0.1 and RC1, respectively). Both of the cell lines were previously selected by continuous exposure to 9-nitro-CPT. The RC0.1 and RC1 cells have high cross-resistance to CPT derivatives including SN-38 and topotecan, but are not cross-resistant to the non-top1 inhibitors etoposide, doxorubicin, and vincristine. Although the top1 protein levels were not decreased in the resistant cells compared with the parental cells, CPT-induced DNA cleavage was markedly reduced in the RC0.1 and RC1 nuclear extracts. The resistant-cell-line nuclear extracts also demonstrated top1 catalytic activity and resistance to CPT, in in vitro assays. Reverse transcription-PCR products from the resistant cell lines were sequenced, and revealed a point mutation resulting in a R364H mutation in the top1 of both RC0.1 and RC1. No wild-type top1 RNA or genomic DNA was detected in the resistant cell lines. Using a purified recombinant R364H top1, we found that the R364H mutant top1 was CPT resistant and fully active. In the published top1 crystal structure, the R364H mutation is close to the catalytic tyrosine and other well-known mutations leading to CPT resistance.

Expression of multidrug resistance associated transporters (MDR1, MRP1, LRP and BCRP) in porcine oocyte.

Transporters such as P-glycoprotein (MDR1), multidrug resistance protein 1 (MRP1), lung resistance-related protein (LRP) and breast cancer resistance protein (BCRP) are associated with multidrug resistance in various carcinoma cell lines. The expression of these molecules has been also characterized in human normal tissues. However, the expression of these molecules in oocyte is still unclear. In order to obtain more insight into the physiological role of these transporters, their expression in porcine oocyte were examined by reverse transcriptase-polymerase chain reaction. MDR1, MRP1 and LRP genes, but not BCRP gene were found to be expressed in porcine oocyte. After the subcloning and sequence analysis of MDR1, MRP1 and LRP genes, the high homology of these transporters were observed between porcine and human gene. These findings suggest that MDR1, MRP1 and LRP play an important physiological role(s) in an oocyte.

Expression of E-cadherin and beta-catenin in primary and peritoneal metastatic ovarian carcinoma.

Protein expression levels of E-cadherin and beta-catenin were examined in 39 primary and 10 metastatic ovarian carcinoma to elucidate the role of these molecules in the extension of ovarian carcinoma by immunohistochemistry. Twenty-two of 39 (56%) ovarian carcinomas were preserved type and 17 of 39 (44%) were reduced type of E-cadherin. In contrast, 36 of 39 (92%) ovarian carcinomas were preserved type and 3 of 39 (8%) were reduced type of beta-catenin. E-cadherin expression in well-differentiated carcinoma was higher than that in moderately/poorly-differentiated carcinoma (p<0.05). Interestingly, 6 of 10 (60%) peritoneal metastatic lesions resulted in the reduced expression of E-cadherin compared with primary lesions. In contrast, only 2 of 10 (20%) metastatic lesions showed reduced expression of beta-catenin compared with primary lesions. Mutation of exon 3 of beta-catenin gene was rare (3%, 1/39) in carcinoma. These results suggested that the cell adhesion molecule E-cadherin might play an important role in the formation of peritoneal metastasis. In contrast, beta-catenin is not a good indicator of metastasis in human ovarian carcinoma.

Use of camptothecin-resistant mammalian cell lines to evaluate the role of topoisomerase I in the antiproliferative activity of the indolocarbazole, NB-506, and its topoisomerase I binding site.

NB-506 is a topoisomerase I (top1) inhibitor in clinical trials. In this study, we used a series of camptothecin (CPT)-resistant cell lines with known top1 alterations. We show that three mutations in different domains of the top1 enzyme that confer CPT resistance also confer cross-resistance to NB-506. The CPT-resistant cell lines and corresponding mutations were: human prostate carcinoma cells DU-145/RC1 (mutation R364H), Chinese hamster fibroblasts DC3F/C10 (mutation G503S), and human leukemia CEM/C2 cells (N722S). This result suggests that NB-506 and CPT share a common binding site in the top1-DNA complex. We next used these three cell lines and their parental cells to study the relationship between top1 poisoning by NB-506 and antiproliferative activity. We found that the CPT-resistant cells were only 2-10-fold resistant to NB-506, which suggests that NB-506 targets other cellular processes/pathways besides top1. This conclusion was further supported by the limited cross-resistance of top1-deficient murine leukemia P388/CPT45 cells (2-fold). Cross-resistance was also limited for J-109,382, an isomer of NB-506 that does not intercalate into DNA, indicating that the non-top1-mediated antiproliferative activity of NB-506 is not attributable to DNA intercalation. Together, these data indicate that NB-506 and indolocarbazoles are promising agents to overcome CPT resistance.

Ecteinascidin 743 induces protein-linked DNA breaks in human colon carcinoma HCT116 cells and is cytotoxic independently of topoisomerase I expression.

Ecteinascidin 743 (Et743; NSC 648766) is a potent antitumor agent presently in clinical trials. Et743 selectively alkylates guanine N2 from the minor groove of duplex DNA and bends the DNA toward the major groove. This differentiates Et743 from other DNA-alkylating agents presently in the clinic. To date, the cellular effects of Et743 have not been elucidated. Recently, Et743 DNA adducts have been found to suppress gene expression selectively and to induce topoisomerase I (top1) cleavage complexes in vitro and top1-DNA complexes in cell culture. In the present study, we characterized the DNA damage and the cell cycle response induced by Et743 in human colon carcinoma HCT116 cells. Alkaline elution experiments demonstrated that micromolar concentrations of Et743 produced comparable frequencies of DNA-protein cross-links and DNA single-strand breaks. The single-strand breaks were protein-cross-linked and were not associated with detectable DNA double-strand breaks. By contrast with camptothecin, these lesions persisted for several hours after drug removal and were not formed at 4 degrees C. Et743 treatment induced transient p53 elevation, dose-dependent cell cycle accumulation in G2-M and in G1- and S-phase, and inhibition of DNA synthesis. The sensitivity of camptothecin-resistant mouse leukemia P388/ CPT45 cells, which fail to express detectable top1, was similar to the sensitivity of wild-type P388 cells, suggesting that top1 is not a critical target for the antiproliferative activity of Et743.

Activity of a novel camptothecin analogue, homocamptothecin, in camptothecin-resistant cell lines with topoisomerase I alterations.

Homocamptothecin (hCPT), which differs from camptothecin (CPT) by the presence of an additional methylene group in the E-ring, was evaluated in CPT-resistant cell lines. Topoisomerase I (top1)-deficient leukemia P388/CPT45 cells were highly resistant to hCPT, which demonstrates that top1 is the primary target of hCPT. Three CPT-resistant cell lines with top1 point mutations (Chinese hamster lung fibroblast DC3F/C10, human prostate carcinoma DU-145/RC1, and human leukemia CEM/C2) and their top1 enzymes were cross-resistant to hCPT. The antiproliferative activity of hCPT was greater than that of CPT in both parental and CPT-resistant cell lines, particularly in the prostate cell lines. The top1 cleavage complexes formed in the presence of hCPT appear to be more stable than those induced by CPT. Together, these data indicate that hCPT is a specific top1 inhibitor, which shares a common binding site with CPT in the topl-DNA cleavage complexes. Because of its potency, hCPT might overcome resistance to CPT in some cancer cells.

Diheteropeptin, a novel substance with TGF-beta-like activity, produced by a fungus, Diheterospora chlamydosporia. II. Physico-chemical properties and structure elucidation.

The structure of diheteropeptin (1), a TGF-beta-like active substance from Diheterospora chlamydosporia Q58044, was determined to be a new cyclotetrapeptide, cyclo[2aminoisobutyryl-(S)-phenylalanyl-(R)-prolyl-(2S,8R,9R)-2-am ino-8,9-dihydroxydecanoyl-] by NMR, mass spectrometric and chemical studies.