Phase II study of transarterial holmium-166-chitosan complex treatment in patients with a single, large hepatocellular carcinoma.

PURPOSE: Holmium-166 ((166)Ho) is a neutron-activated radioactive isotope whose effectiveness in hepatocellular carcinoma (HCC) was first reported in a preclinical study in 1991. Chitosan is a polymer of 2-deoxy-2-amino-D-glucose that readily forms a chelate with heavy metals and converts from a solution under acidic conditions into a gel under neutral or basic conditions. We performed a prospective trial of a transarterial administration of a radiopharmaceutical (166)Ho-chitosan complex in patients with single, large HCC. PATIENTS AND METHODS: The study involved 54 patients who had single HCC (>or=3 cm) without a vascular shunt and were either inoperable or refused surgery. The (166)Ho-chitosan complex was administered at a dose of 20 mCi per cm of tumor diameter (capping at 200 mCi) via the artery that directly fed the tumor. RESULTS: The median tumor size was 5.3 cm (range: 3-13 cm). The response rate was 78% (42/54), and 31 patients had a complete response for a median duration of 27 months. The incidence of grade 3 or 4 leukopenia was 18.6%, anemia 7.4%, thrombocytopenia 27.8%, AST/ALT elevation 26%/24%, and total bilirubin elevation 5.6%. There were two treatment-related deaths (3.7%). Subset analysis revealed a substantial difference between the two groups categorized by tumor size (3-5 vs. >5 cm) with respect to response rate (p = 0.004) and overall survival (p = 0.02). CONCLUSION: We found that transarterial administration of the (166)Ho-chitosan complex was highly effective in the treatment of HCC with acceptable toxicities, especially for patients with tumors of 3-5 cm.

Brain metastases from hepatocellular carcinoma: prognostic factors and outcome: brain metastasis from HCC.

Brain metastases from hepatocellular carcinoma are extremely rare. The objectives of the current study were to assess the natural history, outcome, and possible prognostic factors in patients with brain metastases from hepatocellular carcinoma. Between 1995 and 2006, 6,919 patients with hepatocellular carcinoma were treated at Yonsei University Health System. Of those, 62 (0.9%) had a diagnosis of brain metastasis. We carried out a retrospective review of these 62 patients and performed a statistical analysis. The median age at the time patients were diagnosed with brain metastasis was 54 years. Forty-seven patients (76%) were male, and 53 patients had hepatitis B. Median time from diagnosis of hepatocellular carcinoma to brain metastasis was 18.2 months, and 5 patients had brain involvement as their initial presentation. Intracranial hemorrhage was frequently associated (54.8%) with brain metastasis. The most common presenting symptoms were motor weakness, mental change, and headache. Metastases were treated with whole-brain radiation therapy (WBRT) alone in 17 patients and gamma knife surgery alone in 10 patients. Six patients underwent surgical resection and 5 patients were treated with surgical resection followed by WBRT. Twenty-four patients (39%) received steroids only. Median survival after diagnosis of brain metastasis was 6.8 weeks (95% confidence interval: 3.8-9.8 weeks). Univariate analysis showed that treatment modality, number of brain lesions, alpha-fetoprotein, ECOG performance score, recursive partitioning analysis (RPA) class, and Child-Pugh classification had a statistically significant impact on survival. In multivariate analysis, treatment modality, number of brain lesions, and Child-Pugh classification were statistically significant prognostic factors for survival. The overall prognosis of patients with brain metastases from hepatocellular carcinoma is extremely poor. Nevertheless, some subsets of patients manifested the most favorable survival criteria (single brain metastasis and good liver function); thus, for at least these patients, treatment may result in an improved survival time.

Increments of alpha-dystroglycan expression in liver metastasis correlate with poor survival in gastric cancer.

BACKGROUND: Dystroglycan (DG) is a recently focused adhesion molecule with possible roles in cancer development and progression. We investigated correlations between alpha-DG expression and prognosis in gastric carcinoma with liver metastasis. METHODS: For 40 patients with gastric adenocarcinoma and liver-only metastasis, alpha-DG expression was determined by immunohistochemistry in paraffin-embedded surgical specimens of resected stomach tumor, resected liver metastasis, and their normal counterpart tissues. Correlations between alpha-DG expression and prognosis were retrospectively analyzed. RESULTS: alpha-DG expression was higher in primary gastric cancer (P = 0.006) and lower in liver metastasis (P = 0.002) than in each normal counterpart. In primary stomach cancer, patients who had lower alpha-DG expression in tumors than in normal counterparts showed poor overall survival (OS) (P = 0.028). In contrast, in the liver, patients who had higher alpha-DG expression in tumors than in normal counterparts showed poor OS (P = 0.022). Also, higher alpha-DG expression in liver metastasis than in stomach tumors led to poor recurrence-free survival (P = 0.023) and OS (P = 0.056). CONCLUSIONS: This approach may be used to further understanding of the pathogenesis of liver metastasis from gastric cancer. Further studies are warranted to reveal the mechanisms of alpha-DG dysregulation in liver metastasis.

High-throughput identification of clinically important bacterial pathogens using DNA microarray.

Rapid and accurate detection of pathogenic bacteria is important for the treatment of patients with suitable antibiotics. Here we report the development of a diagnostic DNA microarray for the high-throughput identification of 39 pathogenic bacteria selected based on their high prevalence rate and/or difficulty of cultivation. The 23S ribosomal DNA and 16S-23S rDNA intergenic spacer region were used as target DNAs for pathogen detection. Universal- and species-specific probes were designed based on the unique and common sites within the target DNA sequences. New target DNA sequences were determined for the detection of 19 bacterial pathogens. The usefulness of the designed probes was validated using 39 reference bacteria and also with 515 clinical isolates from various clinical samples including blood, stool, pus, sputum, urine and cerebrospinal fluid. The DNA microarray developed in this study allowed efficient detection of bacterial pathogens with the specificities of 100%. The sensitivities were 100% as well except for the two pathogens, Enterobacter cloacae (75%) and Enterococcus faecium (85%). These results suggest that the DNA microarray-based assay developed in this study outperforms current diagnostic systems with respect to sensitivity, specificity, and high-throughput detection, and thus should be useful in pathogen diagnosis in the clinical setting.

Cytokine expression and cancer detection.

The purpose of the present article is to review the links between cancer and cytokine expression. Cytokines are proteins produced by cells that act as mediators of cell-to-cell communication. Many recent reports indicate that uncontrolled, constitutive cytokine expression in tumors contributes to tumor growth, tumor progression and immuno-suppression, activities that dexceed their usual functions in host anti-tumor response. In addition, cancer susceptibility and severity seem to be associated with functional polymorphisms in cytokine genes. Thus, cytokine expression genomics may have clinical applications. Here we propose approaches to cancer detection in the clinic based on altered patterns of cytokine expression. Although the function and diagnostic importance of cytokines needs to be studied in more detail, examining the relationship between aberrant cytokine expression and cancer promises to be extremely useful for the identification of a new generation of biomarkers, and to advance cancer diagnosis, prevention, and treatment.

Applications of DNA microarray in disease diagnostics.

Rapid and accurate diagnosis of diseases is very important for appropriate treatment of patients. Recent advances in molecular-level interaction and detection technologies are upgrading the clinical diagnostics by providing new ways of diagnosis, with higher speed and accuracy. In particular, DNA microarrays can be efficiently used in clinical diagnostics which span from discovery of diseaserelevant genes to diagnosis using its biomarkers. Diagnostic DNA microarrays have been used for genotyping and determination of disease-relevant genes or agents causing diseases, mutation analysis, screening of single nucleotide polymorphisms (SNPs), detection of chromosome abnormalities, and global determination of posttranslational modification. The performance of DNA-microarray-based diagnosis is continuously improving by the integration of other tools. Thus, DNA microarrays will play a central role in clinical diagnostics and will become a gold standard method for disease diagnosis. In this paper, various applications of DNA microarrays in disease diagnosis are reviewed. Special effort was made to cover the information disclosed in the patents so that recent trends and missing applications can be revealed.

A phase II study of paclitaxel combined with infusional 5-fluorouracil and low-dose leucovorin for advanced gastric cancer.

PURPOSE: The aim of this study was to investigate the efficacy and safety of the combination chemotherapy of paclitaxel, infusional 5-fluorouracil (5-FU) and leucovorin (FLT regimen) in advanced gastric cancer. The primary end point was the time to progression (TTP). METHODS: Patients with evaluable disease with or without measurable lesions received 175 mg/m2 paclitaxel on day 1 followed by 20 mg/m2 leucovorin and 24-h infusion of 5-FU 1,000 mg/m2 (day 1-3) repeated every 3 weeks. RESULTS: Sixty patients were enrolled. The median TTP and overall survival duration were 13 and 60 weeks, respectively. One-year survival rate was 53.3%. Of the 50 patients with measurable lesion, the overall response rate was 31.7%. The most common grade 3-4 adverse event was neutropenia (61.7%). CONCLUSION: The FLT regimen showed an efficacy comparable to other regimens of cisplatin or anthracycline combinations with the advantage of remarkably low non-hematological toxicity. These data about the efficacy of this regimen need confirmation in a phase III trial.

Capecitabine and doxorubicin combination chemotherapy as salvage therapy in pretreated advanced gastric cancer.

PURPOSE: The aim of this study was to evaluate the activity and the safety of a combination regimen of capecitabine and doxorubicin as salvage chemotherapy in advanced gastric cancer patients who had undergone one or two prior chemotherapy regimens. METHODS: Patients received capecitabine, 2,500 mg/m(2)/day PO for 14 days (D1-14) and doxorubicin, 30 mg/m(2) IV on day 1 every 3 weeks until disease progression. The response was evaluated according to RECIST criteria, and the toxicity was evaluated by NCI-CTC (version 2.0). RESULTS: Forty-five patients were enrolled. Twenty-six patients were treated as second-line chemotherapy and the remaining patients as third-line chemotherapy. A total of 152 cycles of chemotherapy (median 2, range 1-12) were administered. Median dose intensities of capecitabine and doxorubicin were 11,326 and 9.6 mg/m(2)/week, respectively. The overall response rate was 6.7% (95% CI, 4.1-12.5%) and the disease control rate was 46.7% (95% CI, 28.6-87.1%) according to an intent-to-treat analysis. The median progression-free survival was 11.3 weeks (95% CI, 5.6-16.7 weeks). The median overall survival was 29.1 weeks (95% CI, 18.3-39.9 weeks) with one-year survival rate of 24%. Severe (grade III/IV) hematologic and non-hematologic toxicity was uncommon and included nausea/vomiting in five (11.1%), neutropenia in two (4.4%), anemia in one (2.2%), and hand-foot syndrome in one patient (2.2%). CONCLUSIONS: The combination of capecitabine and doxorubicin is a feasible salvage regimen in advanced pre-treated gastric cancer.

A single institutional experience of surgically resected thymic epithelial tumors over 10 years: clinical outcomes and clinicopathologic features.

Thymic epithelial tumors (TETs) consist of a series of neoplasm that differ morphologically and biologically. Due to its rarity and indolent natural history, large-scale prospective trials have been lacking. This study aimed to evaluate long-term clinical outcomes and clinicopathologic features for TET after surgical resection and adjuvant treatments. One hundred patients who received surgery plus adjuvant radiotherapy +/- chemotherapy for TET (Masaoka stage II-IVa) from 1995 to 2005 were retrospectively reviewed. Masaoka staging systems were adopted, and pathologic results were classified according to World health organization (WHO) histologic classification. After surgery, 55 patients were treated with radiotherapy alone, while 45 with radiotherapy and chemotherapy. The median radiation dose was 50.4 Gy (45-63 Gy) and six cycles of chemotherapy, consisting of doxorubicin, cisplatin, vincristine and cyclophosphamide, were applied every 3-4 weeks. Distributions according to Masaoka stage were as follows; stage II (58 patients), III (21) and IVa (21). According to WHO histology, there were A (3), AB (7), B1 (7), B2 (31), B3 (31) and C (21). With a median follow-up duration of 65 months (8-143 months), the 5-year overall survival (OS) and disease-free survival (DFS) rates were 75.7% (89.2, 67.9 and 52.1% in stage II, III and IVa, respectively) and 70.3% (83, 62.4 and 33.6% in stage II, III and IVa, respectively). In multivariate analysis, prognostic factors for OS were age, WHO histology, Masaoka stage, and recurrence, while pleural involvement, WHO histology, and Masaoka stage had significant impacts on DFS. Adjuvant chemotherapy did not alter survival outcomes and recurrence patterns. Pleura was the most common recurrence site (15 patients, 53.6%), and significantly associated with pleural recurrence-free survival. In conclusion, pleural involvement at diagnosis was the important prognostic factor, in addition to WHO histology and Masaoka stage. To prevent pleural recurrence and prolong survival, innovative therapeutic approaches warrant further investigations.

Development of a DNA chip for the diagnosis of the most common corneal dystrophies caused by mutations in the betaigh3 gene.

AIM: To develop a diagnostic DNA chip to detect mutations in the betaigh3 gene causing the most common corneal dystrophies (CDs). METHODS: Samples from 98 people, including patients with betaigh3-associated CDs (beta-aCDs), were examined. Specific primer and probe sets were designed to examine exons 4 and 12 of the betaigh3 gene, in order to identify mutant and wild-type alleles. Mutations were then identified by hybridisation signals of sequence-specific probes immobilised on the slide glass. RESULTS: Direct sequencing of exons 4 and 12 of the betaigh3 gene in the patients' genome showed that beta-aCDs could be mainly classified into five types: homozygotic Avellino corneal dystrophy (ACD), heterozygotic ACD, heterozygotic lattice CD I, heterozygotic Reis-Bucklers CD and heterozygotic granular CD. Blind tests were performed by applying the target DNA amplified from the genomic DNA isolated from the peripheral blood of the participants onto a DNA chip. The results obtained by DNA chip hybridisation matched well with the direct DNA sequencing results. CONCLUSIONS: The DNA chip developed in this study allowed successful detection of beta-aCDs with a sensitivity of 100%. Mutational analysis of exons 4 and 12 of the betaigh3 gene, which are the mutational hot spots causing beta-aCDs, can be successfully performed with the DNA chip. Thus, this DNA chip-based method should allow a convenient, yet highly accurate, diagnosis of beta-aCDs, and can be further applied to diagnose other types of CDs.

A pilot study of trans-arterial injection of 166Holmium-Chitosan complex for treatment of small hepatocellular carcinoma.

Percutaneous approaches, such as percutaneous ethanol injection and radiofrequency ablation, have been most widely used for hepatocellular carcinoma patients who were not eligible for surgery. New technologies to improve the efficacy are currently needed. (166)Holmium is a neutron activated radionuclide, and has several beneficial radiophysical characteristics for internal radiation therapy. (166)Holmium-Chitosan complex, in which chitosan is chelated with (166)Holmium, was developed as a radiopharmaceutical for cancer therapy. We have conducted a pilot study to evaluate the clinical efficacy of transarterial administration of (166)Holmium-Chitosan complex in patients with a single and small (< 3 cm) hepatocellular carcinoma. (166)Holmium-Chitosan complex, at a dose of 20 mCi per cm of tumor mass-diameter, was administered through the artery that directly fed the tumor. Twelve patients were treated with a median follow-up duration of 26 (range: 12-61) months. The tumor diameter ranged between 1.5 and 2.5 cm. Ten patients (83%) had complete response and two (17%) had partial response. The median complete response duration was not reached. The median AFP level declined from 83.8 to 8.3 ng/mL within 2 months after treatment. No grade III/IV toxicity was observed. Grade I and II toxicities were observed in four patients (2 abdominal pain, 1 fever, and 1 AST/ALT elevation). No toxic death occurred. This preliminary study shows a promising and durable complete response rate with an acceptable safety profile. Further studies with greater accrual of patients are warranted.

Lack of correlation between P-glycoprotein and chemotherapy resistance in nasal NK/T-cell lymphomas.

Thirty patients with nasal natural killer (NK)/T-cell lymphoma, who underwent systemic chemotherapy with or without involved-field radiotherapy between 1993 and 1998, were retrospectively reviewed to determine the clinical significance of P-glycoprotein immunohistochemically identified in tumor specimens. Eighty percent of previously untreated patients expressed P-glycoprotein. According to P-glycoprotein immunoreactivity, all patients with nasal NK/T-cell lymphoma were divided into 2 groups; (a) P-glycoprotein-negative group (N = 6) and (b) P-glycoprotein-positive group (N = 24). There was no significant difference in clinical profiles between both groups. Regardless of the P-glycoprotein expressions, Epstein-Barr virus genomes were almost identically detected in patients of the 2 groups. Contrary to our expectations, however, P-glycoprotein expressions were not found to be a strong predictor of chemotherapy resistance. Although 2 (33%) of 6 P-glycoprotein-negative patients and 10 (42%) of the 24 P-glycoprotein-positive patients showed a favorable response to systemic chemotherapy, 4 (67%) of 6 P-glycoprotein-negative patients did not achieve complete response (CR) to chemotherapy, which led to an early death, whereas 4 (17%) of the 24 P-glycoprotein-positive patients achieved CR to chemotherapy despite positive P-glycoprotein immunoreactivity. Overall, there were no significant differences in either CR rate or the response rate of patients in the two groups. Overall 5-year actuarial survival and disease-free survival for all patients were 44% and 47%, respectively, but no differences in survival rates were observed between 2 groups. (5-year actuarial survival rate: 33% for the P-glycoprotein-negative, 50% for the P-glycoprotein-positive) (P = 0.7093, log-rank). On univariate and multivariate analyses, P-glycoprotein expressions by immunohistochemical study were not found to be an important prognostic factor. Given these observations, we conclude that the molecular mechanisms of resistance to chemotherapy in nasal NK/T-cell lymphoma patients are not entirely dependent on P-glycoprotein, and that other complex mechanisms of drug action and resistance may be likely to be involved.

Double primary mucoepidermoid carcinoma and hepatocellular carcinoma of the liver--a case report.

Double primary mucoepidermoid-hepatocellular carcinoma of the liver is extremely rare, and only one case has previously been reported in the literature, although there have been about 14 cases of primary mucoepidermoid carcinoma of the liver. Most of the reported hepatic mucoepidermoid carcinoma showed a poor prognosis. We presently report the second case of a double primary mucoepidermoid carcinoma and hepatocellular carcinoma with a brief review of the published literature. A 52-year-old man was admitted because of epigastric pain that lasted for 2 months. A computed tomography of the abdomen revealed a 7-cm, ill-defined mass with irregular marginal enhancement in the left lobe of liver. Another 2-cm nodular tumor was found in segment 8 of the right lobe. The two separate nodules in the patient's liver demonstrated clearly different histologic and immunohistochemical features. The pathological diagnoses were mucoepidermoid carcinoma and hepatocellular carcinoma for the larger and the smaller tumors, respectively. The patient died of liver failure 6 months after a left lobectomy of the liver.

Isolation and characterization of an extracellular antimicrobial protein from Aspergillus oryzae.

A 17 kDa antimicrobial protein was isolated from growth medium containing the filamentous fungus Aspergillus oryzae by extracting the supernatants from the culture media, ion exchange chromatography on CM-sepharose, and C18 reverse-phase high-performance liquid chromatography. This antimicrobial protein, which we considered to be an extracellular antimicrobial protein from A. oryzae (exAP-AO17), possessed antimicrobial activity but lacked hemolytic activity. The exAP-AO17 protein strongly inhibited pathogenic microbial strains, including pathogenic fungi, Fusarium moniliform var. subglutinans and Colletotrichum coccodes, and showed antibacterial activity against bacteria, including E. coli O157 and Staphylococcus aureus. To confirm that the protein acts as a regulation factor for extracellular secretion, we examined growth under varying conditions of N sources, C sources, ions, ambient pH, and stress. Various culture conditions were found to induce characteristic changes in the expression of protein synthesis as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Highly basic polypeptides were regulated by suppressing the ambient pH under acidic conditions and strongly induced under alkaline conditions, thus confirming that pH regulation is physiologically relevant. The expression of exAP-AO17 was upregulated by heat shock upon growth in the presence of NaCl. Automated Edman degradation showed that the N-terminal sequence of exAP-AO17 was NH 2-GLPGPAGAVGFAGKDQNM-. ExAP-AO17 showed partial sequence homology with a collagen belonging to the animal source. These results suggest that exAP-AO17 is an excellent candidate as a lead compound for the development of novel oral or other types of anti-infective agents.

Mobilized CD34+ cells as a biomarker candidate for the efficacy of combined maximal tolerance dose and continuous infusional chemotherapy and G-CSF surge in gastric cancer.

We investigated whether the level of bone marrow-derived progenitor cells and mature endothelial cells could be used as predictors of clinical outcome in patients receiving taxotere-based chemotherapy for advanced gastric cancer. Peripheral blood mononuclear cells were obtained from 49 gastric cancer patients who received taxotere combined with 5-FU and leucovorin and prophylactic G-CSF treatment. To categorize the cells, the cell markers CD34, vWF, P1H12, and CD31 were stained. Changes in these cells were examined before and after chemotherapy, and the clinical significance of these changes to response prediction and prognosis were investigated. Before the second cycle of chemotherapy, the number of CD34+/vWF+ and CD34+ cells was higher in non-responders as compared to the responders. Patients with > or =6.2 CD34+/vWF+ cells/ml had a shorter progression free survival (3.7 months) as against patients with <6.2 CD34+/vWF+/ml (6.0 months, p = 0.076). Patients with > or =5.8 CD34+ cells/ml had shorter progression free survival (4.0 months) than patients with <5.8 CD34+ cells/ml (6.1 months, p = 0.046). In an ex vivo pharmacokinetic study, the maximum inhibition (I(max)) for HUVEC and YCC3 cells was 13.0 +/- 6.6% and 74.0 +/- 2.0%, respectively. The time to reach I(max) (T(max)) was 72 h in all HUVEC cells and 0.5 hours in YCC3 cells. We suggested that CD34+/vWF+ and CD34+ cells can be used as a biomarker for prediction and CD34+ cells for prognosis.

Changing patterns of prognosticators during 15-year follow-up of advanced gastric cancer after radical gastrectomy and adjuvant chemotherapy: a 15-year follow-up study at a single korean institute.

BACKGROUND: We evaluated the long-term natural history of gastric cancer after radical gastrectomy and adjuvant chemotherapy through a 15-year follow-up study at a single institute. METHODS: Five hundred patients with advanced gastric adenocarcinoma who received radical gastrectomy and adjuvant chemotherapy were included in this long-term follow-up study. Patients were evaluated by imaging studies and upper gastrointestinal series or endoscopy every 6 months until the 10th year after surgery. Since then, the patients have been followed yearly in the same manner. RESULTS: The median follow-up period was 190.5 months. The recurrence rate in 5-year survivors was 10.8%. The dominant recurrence pattern was peritoneal carcinomatosis within 5 years and distant metastasis after 5 years post gastrectomy. Tumor stage was a clear-cut prognosticator within 5 years post gastrectomy, but was no longer informative in 5-10 years. At this period, only stage IV (IB-IIIB vs IVM0) was a significantly poor prognosticator. After 10 years, second primary cancer (seven cases) became as important an issue as recurrence of primary gastric cancer (six cases). CONCLUSIONS: In patients with gastric carcinoma treated with radical gastrectomy and adjuvant chemotherapy, late recurrence after 5 years post gastrectomy was not rare. Prognosticators were varied depending on the length of time after surgery. Tumor factors including stage were prognosticators within 5 years post gastrectomy, but tumor factors except stage IV had no prognostic value after 5 years. In the 5-10 years post gastrectomy, only stage IV (IB-IIIB vs IVM0) was a poor prognosticator. Also, after 10 years, there were no prognosticators.

DNA microarray-based detection of nosocomial pathogenic Pseudomonas aeruginosa and Acinetobacter baumannii.

Infection by nosocomial pathogenic bacteria is increasingly becoming a major threat to the patients in the hospital. We have developed a diagnostic DNA microarray for the detection of two important nosocomial pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii. The diagnostic DNA microarray contains the species-specific probes of 15mer oligonucleotides designed based on the sequences of 23S ribosomal DNA. The performance of DNA microarray in diagnosing P. aeruginosa and A. baumannii was evaluated using reference bacteria as well as clinical specimens such as blood, stool, pus, sputum, urine and cerebrospinal fluid. Using this DNA microarray, A. baumannii could be successfully detected in 11 out of 13 clinical specimens, thus giving the sensitivity of 84.6% with the specificity of 100% and the positive predictive value of 100%. P. aeruginosa could also be detected in 25 out of 26 clinical specimens, showing the sensitivity of 96.2%, the specificity of 100%, and the positive predictive value of 100%. These results suggest that two nosocomial pathogens, P. aeruginosa and A. baumannii, can be efficiently diagnosed by using the DNA microarray developed in this study.

Constitutive production of human leptin by fed-batch culture of recombinant rpoS- Escherichia coli.

High-level production of human leptin by fed-batch culture of recombinant Escherichia coli using constitutive promoter system was investigated. For the constitutive expression of the obese gene encoding human leptin, the strong constitutive HCE promoter cloned from the D-amino acid aminotransferase gene of Geobacillus toebii was used. To develop an optimal host-vector system, several different recombinant E. coli strains were compared for leptin production. In flask cultures, E. coli FMJ123, which is a rpoS mutant strain, showed the highest level of leptin production (41% of total proteins). By comparing the expression levels of leptin in several different rpoS- and rpoS+ strains, it could be concluded that rpoS mutation positively affected constitutive production of leptin. For the large-scale production of human leptin, fed-batch cultures of recombinant E. coli FMJ123 were carried out using three different feeding solutions--chemically defined, yeast extract-containing, and casamino acid-containing feeding solutions. Among these, the use of casamino acid-containing feeding solution allowed production of leptin up to 2.1 g/L, which was 2.1- and 1.8-fold higher than that obtained with chemically defined and yeast extract-contained feeding solutions, respectively. These results suggest that the HCE promoter can be used for the efficient production of leptin, and most likely other recombinant proteins, in a constitutive manner.

Changes of Telomerase Activity and Proliferation by Inhibition of Reverse Transcriptase Activity in Human Cancer Cell.

PURPOSE: Activation of telomerase is proposed to be an essential step in cancer cell immortalization and cancer progression. 3'-azido-2',3'-dideoxythymidine (AZT), a reverse transcriptase inhibitor, was reported to be incorporated in telomeric sequences of immortalized cells in culture and to suppress the activity of telomerase and the cell proliferation. In this study, after induction of cancer cell senescence with long-term treatment of AZT, we investigated the dynamics of telomerase subunits (hTERT, hTR, TEP), transcription factors (c-Myc, Mad1), telomerase activity, and finally, telomere length in a human breast cancer cell line. MATERIALS AND METGODS: Human breast cancer cell (MDA-MB-231) was treated with AZT. Senescence was measured by senescence-associated beta-gal staining and apoptosis was counted by dTd enzyme assay. Telomerase activity (by TRAP assay), expression of telomerase subunit genes (by RT-PCR and real-time PCR) and telomere length (by Southern blot analysis) were measured after the AZT treatment. RESULTS: We found evidences of senescence, apoptosis and growth delay after AZT treatment. In addition, AZT- treated cancer cells showed inhibition of telomerase activity and shortening of telomere length in a dose- and duration-dependent way. Among the telomerase subunits, hTERT and c-Myc were the first factors to change after AZT treatment, subsequently, followed by the changes of hTR, Mad1 and TEP. CONCLUSION: The suppression of hTERT and c-Myc by AZT treatment was the initial genetic phenomenon, subsequently followed by the changes of hTR, Mad1 and TEP.

Paclitaxel and Cisplatin Combination Chemotherapy in Pretreated Breast Cancer.

PURPOSE: A single institute trial of combination chemotherapy, with paclitaxel and cisplatin, in patients with metastatic breast cancer, having failed previous combination chemotherapy, was performed. MATERIALS AND METHODS: Patients were only eligible for this study if there disease had progressed, following treatment with previous chemotherapy, in either an adjuvant or a metastatic setting. Paclitaxel 175 mg/m2 was administered as a 3-hour continuous infusion on day 1, and cisplatin 80 mg/m2 was administered for 2 hours on day 2, with adequate hydration. This was repeated every 3 weeks, and continued until one of the following events occurred: disease progression, unacceptable adverse effect or treatment refusal by the patient. Intercurrent palliative radiotherapy, or concurrent hormonal therapy, was permitted, depending on each patient's status. All the endpoints were evaluated under the principle of intention to treat analysis. RESULTS: A total of 24 patients entered the study, and 18 had at least one measurable lesion, but 6 did not. The objective response rate of the 18 patients was 50%(9/18). Two were complete responses and seven showed partial responses. The median response duration, progression free and overall survival were 5.3 months (range, 4~18), 6 months (95% CI, 5~7) and 12 months (95% CI, 7~17), respectively. 67% of the planned dose was administered. Out of a total 135 cycles administered, about 20% of cycles showed grade 3 or 4 leukopenia and 7% showed grade 3 thrombocytopenia. Two patients suffered from pneumonia, and one experienced neutropenic fever. Mucositis, greater than grade 3, existed in three cases. No treatment related deaths were reported. CONCLUSION: The combination chemotherapy, with paclitaxel and cisplatin, was active in the treatment of metastatic breast cancer patients having failed previous chemotherapy.