PURPOSE: When murine myeloma cells P3-X63-Ag8.653 (V653) of this model treated with amin opterin, an anticancer drug, they can't synthesize nucleic acid via de novo or salvage pathway and selectively eliminated due to apoptosis. This study was aimed to clone specific known and novel genes preferentially expressed in aminopteirn-treated tumor cell apoptosis. MATERIALS AND METHODS: This study was aimed to clone specific known and novel genes pre ferentially expressed in aminopteirn-treated tumor cell apoptosis by using subtraction-PCR technique. RESULTS: By using this technique 868 clones were obtained. Of these 427 clones were positive with insert DNA. By using cross-hybridization Southern blotting, final 101 clones were selected. All of these genes were sequenced and analyzed by using genebank DNA database. Total 101 clones of genes preferentially expressed in apoptotic tumor cells were classified into 10 groups, which included ribosomal proteins, nuclear proteins, mitdegrees Chondrial proteins, signal transductional proteins, retroviral proteins, cell surface receptor proteins, cell structural proteins, unclassified miscellaneous proteins, and novel genes. Especially, Unknown novel genes preferentially ex pressed in this apoptotic tumor cells included clone numbers S1-63, 1-1, 1-3, 1-16, 1-18, 1-20, 3-33, 3-41, 3-44, 3-48, 3-55, 3-60, 6-17, 6-25, 8-12, 50-7, 50-23, and 100-35. CONCLUSION: It seemed that known and unknown novel genes cloned in this study would con tribute to the future studies regarding apoptosis of tumor cells and cancer treatment therepy.
Aminopterin ; Apoptosis* ; Blotting, Southern ; Clone Cells* ; Cloning, Organism* ; Databases, Nucleic Acid ; DNA ; Mass Screening* ; Membrane Proteins ; Nuclear Proteins ; Ribosomal Proteins ; Zidovudine

PURPOSE: Cryopreservation has been the standard method of storing hematopoietic cells for the past 20 years, but this prdegrees Cedure is laborious and expensive. So, we evaluated the hematopoietic recovery of stored PBSCs at 4degrees C for a variable storage period MATERIALS AND METHODS: Eight leukapheresis products were kept unprdegrees Cessed at 4degrees C for 96 hours. To evaluate the effect of storage period on the hematopoietic recovery of PBSCs, assays for viability of mononuclear cells (MNCs), CFU-GM colony counts and CD34 cell counts were performed every 24 hours after PBSC collection. We tried to compare hematopoetic recovery of stored PBSCs at 4degrees C with that of cryopreserved PBSCs by using repeated measures ANOVA. RESULTS: Viability of MNCs, CFU-GM colony counts and CD34 cell counts were monitored at 24 hour, 48 hour, 72 hour and 96 hour after PBSC collection. Data are expressed as percentage of baseline value and shown as mean s.d.; MNCs viability (96+/-2%, 94+/-2%, 92+/-2%, 88+/- 3%), CFU-GM colony counts (87+/-10%, 79+/-11%, 65+/-13%, 56+/-15%), and CD34 cell counts (93+/-13%, 93+/-12%, 88+/-14%, 85+/-19%). After storing PBSCs at 4degrees C for 96 hours, viability of MNCs and CFU-GM colony counts were significantly reduced (p<0.05) except CD34 cell concentration (p>0.05). Prdegrees Cedures of controlled-rate freezing and thawing resulted in a notable loss of viability (77+/-9%) and CFU-GM colony count (71+/-29%). CFU-GM colony counts of 72 hour-stored PBSCs at 4degrees C was similar to those of cryopreserved PBSCs. CONCLUSION: If G-CSF mobilized PBSCs are stored at 4degrees C in less than 72 hours after collection, those hematopoietic recovery would be comparable to that of cryopreserved stem cells which are achieved by the rate-control freezer.
Bezafibrate ; Cell Count ; Cryopreservation ; Freezing ; Granulocyte Colony-Stimulating Factor ; Granulocyte-Macrophage Progenitor Cells ; Leukapheresis ; Stem Cells*

PURPOSE: The purpose was to investigate the spontaneous apoptotic index (SAI) and p53 protein expression and to identify the role of SAI and p53 protein positivity. MATERIALS AND METHODS: Forty six patients with squamous cell carcinoma of the cervix, FIGO stage IIB, treated with curative radiotherapy alone between 1990 and 1993 were included in this study. Definitive radiotherapy including external beam and high-dose-rate brachytherapy was given. Pretreatment paraffin-embedded biopsy specimens of those patients were scored for apoptosis and p53 protein expression using mouse mondegrees Clonal antibody (DO-7) by immuno staining. Clinicopathologic characteristics were also studied in relation to SAI and p53 protein expression, and as prognostic factors for clinical outcome. RESULTS: SAI and p53 were not related to any clinical characteristics. The range of the SAI was 0.2~4.7% (median 1.1%, mean 1.5%). The rate of p53 protein expression was 65.2% (30/46). Patients whose tumors had high SAI and low p53 protein positivity had better treatment outcome than those with lower SAI. There was also a significant correlation between the SAI and p53 protein expression. CONCLUSION: The pretreatment SAI and p53 oncoprotein expression are clinically useful in predicting the clinical outcome of FIGO stage IIB squamous cell carcinoma of the uterine cervix patients treated with definitive radiotherapy.
Animals ; Apoptosis* ; Biopsy ; Brachytherapy ; Carcinoma, Squamous Cell* ; Cervix Uteri* ; Female ; Humans ; Mice ; Radiotherapy* ; Treatment Outcome ; Uterine Cervical Neoplasms

No abstract available.
Humans* ; Metalloproteases ; Prostate* ; Prostatic Neoplasms

PURPOSE: The biologic behavior of tumor cells is partially controlled by the microenvironment. We investigated the expression levels of several genes involved in metastasis and drug response in RENCA cells growing in ectopic (skin) and orthotopic (kidney) sites. MATERIALS AND METHODS: Murine renal carcinoma cells were injected into kidney (orthotopic) and subcutis (ectopic) of syngeneic mice. Mice were treated with doxorubicin (DXR) (8 mg/kg) on days 8 and 15 after tumor cell implantation. Drug response was measured both in vivo and ex vivo by measuring tumor size and MTT assay. We also performed an in situ mRNA hybridization to estimate the expression levels of mdr (multidrug resistance), EGFR (epidermal growth factor receptor) and type IV collagenase. RESULTS: RENCA cells growing in the kidney of syngeneic mice produced metastatic lesions in the lung (57% of mice), while the same cells growing in the subcutis did not. Tumors growing in the kidney were more resistant to DXR than tumors growing in the subcutis. MTT assays revealed that tumor cells derived from kidney were more resistant to DXR than those cells from subcutis. In situ hybridization analyses showed that transcripts of EGFR and type IV collagenase genes in kidney tumors were higher than those of subcutaneous tumors but mdr expression showed no difference between the two tumors. CONCLUSION: These results demonstrate that the organ environment influences the drug responsive ness and the expression of EGFR and type IV collagenase genes in murine renal cell carcinoma cells.
Animals ; Carcinoma, Renal Cell* ; Collagenases ; Doxorubicin ; Gene Expression Regulation* ; In Situ Hybridization ; Kidney ; Lung ; Mice* ; Neoplasm Metastasis ; Receptor, Epidermal Growth Factor ; RNA, Messenger

PURPOSE: To evaluate the efficacy and toxicity of combination chemotherapy with vinorelbine, ifosfamide, and cisplatin in patients with advanced non-small cell lung cancer. MATERIALS AND METHODS: Patients with unresectable, pathologically proven non-small cell lung cancer who had no prior chemotherapy were eligible. Patients received vinorelbine (25 mg/m2, iv., D1 & 8), ifosfamide (1.5 g/m2, iv., D1-3 with mesna), and cisplatin (60 mg/m2, iv., D1). The treatment was repeated every 3 weeks. RESULTS: Between degrees Ctober, 1997 and June, 1999, 26 patients were enrolled. Median age was 61. 1 patient had stage IIIA, 13 had stage IIIB, and 12 had stage IV. Patients with adendegrees Carcinoma were 15, squamous cell carcinoma were 11. Of 22 evaluable patients, objective responses were observed in 9 patients (response rate: 40.9%, CR: 1 (4.5%), PR 8 (36.4%)). Median duration of response was 48 weeks. Median overall survival was 52 weeks. Grade 3-4 leukopenia was observed in 10.2% of the 88 courses. There was 1 death related to febrile neutropenia. Non- hematologic toxicities were mild. CONCLUSION: We concluded that combination chemotherapy with vinorelbine, ifosfamide, and cisplatin was effective and tolerable in patients with advanced non-small cell lung cancer, and phase III randomized trial is needed to compare this regimen to other cisplatin-based regimens.
Carcinoma, Non-Small-Cell Lung* ; Carcinoma, Squamous Cell ; Cisplatin* ; Drug Therapy ; Drug Therapy, Combination* ; Febrile Neutropenia ; Humans ; Ifosfamide* ; Leukopenia

PURPOSE: Chromosome microdissection has been recommended as a technology to overcome the limited problems of conventional cytogenetic analysis and is a direct approach to isolate DNA from specific interesting region of chromosome. KUMA-1 cell line has a specific reserved chromosome abnormality during prdegrees Cess from primary cancer culture to continuous cell line development, der(2)t(2;?)(qter;?). So molecular analysis for transldegrees Cation region of der(2) may be helpful to understand pathogenesis of this primary cancer. The aim of this study was to develop painting probe for the transldegrees Cation region for molecular study in future about transldegrees Cation region of der(2) of KUMA-1 cell line. MATERIALS AND METHODS: KUMA-1 cell line was derived from a squamous cell carcinoma of urinary bladder. The transldegrees Cation breakpoint region of der(2) appeared in KUMA-1 cell line was microdissected and dissected chromosome segments were amplified by PCR reaction. Fluorescent in situ hybridization was conducted on KUMA-1 metaphase cells with the probe generated from PCR product to confirm the construction of painting probe containing the transldegrees Cation breakpoint of der(2). RESULTS: Painting probe was hybridized to the metaphase chromosome of KUMA-1 cell line and two fluorescent signals were mapped to the transldegrees Cation forming chromosomal region of der(2). CONCLUSION: It was possible to construct the painting probe for the transldegrees Cation region of der (2) by chromosome microdissection.
Carcinoma, Squamous Cell ; Cell Line ; Chromosome Aberrations ; Cytogenetic Analysis ; DNA* ; In Situ Hybridization, Fluorescence ; Metaphase ; Microdissection* ; Paint* ; Paintings* ; Polymerase Chain Reaction ; Urinary Bladder

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent angiogenic factor of many solid tumors, promoting vascularization and formation of metastases. In an attempt to generate effective VEGF inhibitors, the authors constructed the VEGF receptor mutants, expressed in E. coli and Sf9 insect cells, and examined their binding to VEGF. MATERIALS AND METHODS: The cDNA fragment encoding FLT-1 extracellular domain was cloned from human umbilical vein endothelial (HUVE) cell total RNA using RT-PCR. PCR- subcloning was performed using this template, in order to generate the deletion mutants by introducing FLT-1 partial sequences into E.coli expression vector pET-21d and baculovirus transfer vactors, pBAC-1 and pBAC-3. Two mutant proteins from baculovirus-infected insect cells were purified by heparin sepharose chromatography and immobilized into nitrdegrees Cellulose membrane followed by 125I-VEGF binding assay. RESULTS: Two mutant receptors, sFLT (1~7) and sFLT (2~4) expressed in E.coli appeared in inclusion body as insoluble proteins. The soluble mutant receptors were produced in low yield by baculovirus/insect cell expression system. Both immobilized mutant receptors, sFLT (1~7) and sFLT (2~4) were able to bind VEGF. CONCLUSION: These results suggest that a small soluble mutant receptor, sFLT (2~4), as well as sFLT (1~7) may be used effectively for bldegrees Cking angiogenic function of VEGF.
Angiogenesis Inducing Agents ; Baculoviridae ; Cellulose ; Chromatography, Agarose ; Clone Cells ; DNA, Complementary ; Heparin ; Humans ; Inclusion Bodies ; Insects ; Membranes ; Mutant Proteins ; Neoplasm Metastasis ; Protein-Tyrosine Kinases ; Receptors, Vascular Endothelial Growth Factor* ; RNA ; Umbilical Veins ; Vascular Endothelial Growth Factor A*

No abstract available.
Hyperplasia*

PURPOSE: Cell-cell interactions determine normal prostate development and subsequent neoplastic transfor mation. The progression of prostate cancer from androgen-dependent to androgen-independent states involves multiple steps of genetic changes mediated by tumor-microenvironment interactions. To understand the epigenetic factors that lead to progression, we studied if 1) androgen-dependent and non-metastatic LNCaP may interact with prostate or bone fibroblasts under microgravity-simulated conditions in vitro. 2) LNCaP may interact with prostate fibroblasts in vivo, and acquire androgen-independence and metastatic potential. MATERIALS AND METHODS: The LNCaP sublines were generated as follows. 1) LNCaP cells were grown in vitro either alone or with prostate or bone fibroblasts under microgravity-simulated conditions. 2) LNCaP cells were grown in vivo as chimeric tumors with prostate fibroblasts. The LNCaP sublines were characterized by studies of chromosomal analysis, comparative genomic hybridization and, in vivo tumorigenicity and metastatic potential. RESULTS: In comparison to the parental LNCaP cells, the LNCaP sublines underwent permanent genotypic and phenotypic changes manifested in androgen-independence and metastatic potential. CONCLUSION: These results emphasize the importance of cell-cell interaction as a critical determinant that could "induce" or "select" progenies favoring enhanced prostate cancer growth and progression. This concept favors the development of toxic gene therapy targeting both prostate cancer epithelium and supporting bone stroma for an effective eradication and prevention of prostate cancer bone metastasis.
Cell Line* ; Comparative Genomic Hybridization ; Epigenomics ; Epithelium ; Fibroblasts* ; Genetic Therapy ; Humans ; Neoplasm Metastasis ; Parents ; Prostate* ; Prostatic Neoplasms*