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Biomedical subjects

W Franklin

Publications and source records attributed to W Franklin.

At least 19 recordsLinked to original sources
PubMed2000

Altered HOX and WNT7A expression in human lung cancer.

HOX genes encode transcription factors that control patterning and cell fates. Alterations in HOX expression have been clearly implicated in leukemia, but their role in most other malignant diseases remains unknown. By using degenerate reverse transcription-PCR and subsequent real-time quantitative assays, we examined HOX expression in lung cancer cell lines, direct tumor-control pairs, and bronchial epithelial cultures. As in leukemia, genes of the HOX9 paralogous group and HOXA10 were frequently overexpressed. For HOXB9, we confirmed that elevated RNA was associated with protein overexpression. In some cases, marked HOX overexpression was associated with elevated FGF10 and FGF17. During development, the WNT pathway affects cell fate, polarity, and proliferation, and WNT7a has been implicated in the maintenance of HOX expression. In contrast to normal lung and mortal short-term bronchial epithelial cultures, WNT7a was frequently reduced or absent in lung cancers. In immortalized bronchial epithelial cells, WNT7a was lost concomitantly with HOXA1, and a statistically significant correlation between the expression of both genes was observed in lung cancer cell lines. Furthermore, we identified a homozygous deletion of beta-catenin in the mesothelioma, NCI-H28, associated with reduced WNT7a and the lowest overall cell line expression of HOXA1, HOXA7, HOXA9, and HOXA10, whereas HOXB9 levels were unaffected. Of note, both WNT7a and beta-catenin are encoded on chromosome 3p, which undergoes frequent loss of heterozygosity in these tumors. Our results suggest that alterations in regulatory circuits involving HOX, WNT, and possibly fibroblast growth factor pathways occur frequently in lung cancer.

Blotting, Western↗
PubMed2000

Ex vivo expanded peripheral blood progenitor cells provide rapid neutrophil recovery after high-dose chemotherapy in patients with breast cancer.

Ex vivo expanded peripheral blood progenitor cells (PBPCs) have been proposed as a source of hematopoietic support to decrease or eliminate the period of neutropenia after high-dose chemotherapy. CD34 cells were selected from rhG-CSF mobilized PBPCs from patients with breast cancer and were cultured for 10 days in defined media containing 100 ng/mL each of rhSCF, rhG-CSF, and PEG-rhMGDF in 1 L Teflon bags at 20 000 cells/mL. After culture the cells were washed and reinfused on day 0 of transplantation. On day +1, cohort 1 patients (n = 10) also received an unexpanded CD34-selected PBPC product. These patients engrafted neutrophils (absolute neutrophil count, >500/microL) in a median of 6 (range, 5-14) days. Cohort 2 patients (n = 11), who received expanded PBPCs only, engrafted neutrophils in a median of 8 (range, 4-16) days. In comparison, the median time to neutrophil engraftment in a historical control group of patients (n = 100) was 9 days (range, 7-30 days). All surviving patients are now past the 15-month posttransplantation stage with no evidence of late graft failure. The total number of nucleated cells harvested after expansion culture was shown to be the best predictor of time to neutrophil engraftment, with all patients receiving more than 4 x 10(7) cells/kg, engrafting neutrophils by day 8. No significant effect on platelet recovery was observed in any patient. These data demonstrate that PBPCs expanded under the conditions defined can shorten the time to engraftment of neutrophils compared with historical controls and that the rate of engraftment is related to the dose of expanded cells transplanted.

Antigens, CD↗
PubMed2000

Autologous transplantation of ex vivo expanded bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer.

The collection of small aliquots of bone marrow (BM), followed by ex vivo expansion for autologous transplantation may be less morbid, and more cost-effective, than typical BM or blood stem cell harvesting. Passive elimination of contaminating tumor cells during expansion could reduce reinoculation risks. Nineteen breast cancer patients underwent autotransplants exclusively using ex vivo expanded small aliquot BM cells (900-1200 x 10(6)). BM was expanded in media containing recombinant flt3 ligand, erythropoietin, and PIXY321, using stromal-based perfusion bioreactors for 12 days, and infused after high-dose chemotherapy. Correlations between cell dose and engraftment times were determined, and immunocytochemical tumor cell assays were performed before and after expansion. The median volume of BM expanded was 36.7 mL (range 15.8-87.0). Engraftment of neutrophils greater than 500/microL and platelets greater than 20,000/microL were 16 (13-24) and 24 (19-45) days, respectively; 1 patient had delayed platelet engraftment, even after infusion of back-up BM. Hematopoiesis is maintained at 24 months, despite posttransplant radiotherapy in 18 of the 19 patients. Transplanted CD34(+)/Lin(-) (lineage negative) cell dose correlated with neutrophil and platelet engraftment, with patients receiving greater than 2.0 x 10(5) CD34(+)/Lin(-) cells per kilogram, engrafting by day 28. Tumor cells were observed in 1 of the 19 patients before expansion, and in none of the 19 patients after expansion. It is feasible to perform autotransplants solely with BM cells grown ex vivo in perfusion bioreactors from a small aliquot. Engraftment times are similar to those of a typical 1000 to 1500 mL BM autotransplant. If verified, this procedure could reduce the risk of tumor cell reinoculation with autotransplants and may be valuable in settings in which small stem cell doses are available, eg, cord blood transplants. (Blood. 2000;95:2169-2174)

Adult↗
PubMed2000

Adult-type pulmonary function tests in infants without respiratory disease.

A new method that permits the measurement of adult-type maximal expiratory flow-volume curves and fractional lung volumes in sedated infants was recently described. The purpose of this study was to define the normal range for these new measures of pulmonary function in infants and young children. Measurements of forced expiratory flows and fractional lung volume were made on 35 occasions in 22 children (ages 3-120 weeks) without respiratory disease. Maximal expiratory flow-volume curves were measured by the raised lung volume, thoracoabdominal compression technique. Functional residual capacity (FRC) was measured plethysmographically. Measurements of total lung capacity (TLC), residual volume (RV), FRC, forced vital capacity (FVC), and forced expiratory flows at 25, 50, 75, 85, and between 25% and 75% of expired FVC (FEF(25), FEF(50), FEF(75), FEF(85), and FEF(25-75), respectively) all increased in relation to infant length (P<0.001). RV/TLC, FRC/TLC, and FEF(25-75)/FVC declined in relation to increasing length (P<0.001). The forced expiratory flow and fractional lung volume measurements using this method were similar to previously reported estimates using other methods. These estimates represent a reasonable reference standard for infants and young children with respiratory problems.

Anesthesia, General↗
PubMed2000

HER-2/neu as a therapeutic target in non-small cell lung cancer, prostate cancer, and ovarian cancer.

HER-2/neu is overexpressed in most epithelial malignancies. Lung cancer, prostate cancer, and ovarian cancer are common epithelial tumors in which clinical trials are currently in progress to explore the potential therapeutic role for monoclonal antibodies to HER-2/neu (trastuzumab [Herceptin; Genentech, Inc, South San Francisco, CA]). In preclinical studies with tumor cell lines, trastuzumab was found to have additive and synergistic effects with some chemotherapeutic agents. Clinical trials investigating combination chemotherapy with trastuzumab and a variety of chemotherapeutic agents are already in progress in lung cancer.

Animals↗
PubMed1999

Ex-vivo expansion of bone marrow progenitor cells for hematopoietic reconstitution following high-dose chemotherapy for breast cancer.

The use of hematopoietic growth factors, stromal monolayers, and frequent medium exchange allows the expansion of hematopoietic progenitors ex-vivo. We evaluated the use of ex-vivo expanded progenitor cells for hematopoietic reconstitution following high dose chemotherapy (HDC) in breast cancer patients. Patients with high-risk Stage II or metastatic breast carcinoma underwent bone marrow aspirations using general anesthesia. A total of 675-1125 x 10(6) mononuclear cells (MNC) were seeded for ex-vivo expansion for 12 days in controlled perfusion bioreactors (Aastrom Biosciences, Inc.). The bone marrow cultures, which included the stromal cells collected with the aspirate, were supplemented with erythropoietin, granulocyte-macrophage-colony stimulating factor (GM-CSF)/IL-3 fusion protein (PIXY 321), and flt3 ligand. Stem cell transplant was performed with expanded cells after HDC. A median bone marrow volume of 52.9 mL (range 42-187 mL) was needed to inoculate the bioreactors. Median fold expansion of nucleated cells (NC) and colony forming unit granulocyte-macrophage (CFU-GM) was 4.9 and 9.5, respectively. The median fold expansion of CD34+lin- and long-term culture-initiating culture (LTC-IC) was 0.42 and 0.32, respectively. Five patients were transplanted with ex-vivo expanded NC. Median days to an absolute neutrophil count > 500/microL was 18 (range 15-22). Median days to a platelet count > 20,000/microl was 23 (range 19-39). All patients had sustained engraftment of both neutrophils and platelets. Immune reconstitution was similar to that seen after HDC and conventional stem cell transplantation. We conclude that ex-vivo expansion of progenitor cells from perfusion cultures of small volume bone marrow aspirates, allows hematopoietic reconstitution after HDC.

Adult↗
PubMed1998

PTEN/MMAC1 mutations identified in small cell, but not in non-small cell lung cancers.

A putative tumor suppressor, PTEN/MMAC1 gene at 10q23 was recently identified and found to be mutated in many different human tumors. To determine the role of the PTEN/MMAC1 gene in lung cancer, we screened 34 small cell lung cancer (SCLC) cell lines, 10 SCLC tumors, 13 non-small cell lung cancer (NSCLC) cell lines and 10 NSCLC tumors using Denaturing HPLC (DHPLC) and direct sequencing methods. In SCLC, six (18%) of the cell lines and one of the primary tumor samples (10%) showed alterations of the PTEN/MMAC1 gene including point mutations, small fragment deletions, and homozygous deletions. All of the point mutations and small fragment deletions were observed in hemizygously deleted cell lines. In contrast to SCLC, none of the NSCLC tumors or cell lines had mutations in the PTEN/MMAC1 gene. These data indicate that PTEN/MMAC1 mutations contribute to the pathogenesis and neoplastic evolution in SCLC but not in NSCLC.

Carcinoma, Non-Small-Cell Lung↗
PubMed1997

Ex vivo expansion of megakaryocyte progenitors: effect of various growth factor combinations on CD34+ progenitor cells from bone marrow and G-CSF-mobilized peripheral blood.

Prolonged thrombocytopenia resulting from inadequate megakaryocyte (MK) progenitor cell reconstitution is a serious complication of hematopoietic cell-supported high-dose chemotherapy (HDC). In this situation, the infusion of MK progenitors that are expanded ex vivo could be clinically beneficial. In this study we investigated the ability of various growth factor combinations to generate MK progenitors. CD34+ cells derived from bone marrow (BM) and granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (PB) from 17 patients with breast cancer, lymphoma, or myeloma were cultured unpertubed for 10 days in a serum-free liquid culture system that contained recombinant growth factors. Five different growth factors combinations were evaluated: Stem cell factor (SCF), interleukin (IL)-3, IL-6 + G-CSF (combination 1); SCF, megakaryocyte growth and development factor (MGDF) + G-CSF (combination 2); SCF + MGDF (combination 3); MGDF alone (combination 4); and SCF, IL-3, IL-6, G-CSF + MGDF (combination 5). PB CD34+ cells yielded significantly higher numbers of CD41+ MK progenitors than BM CD34+ cells with any of the growth factor regimens assayed. PB CD34+ cells (2x10[5]) at day 0 generated 1.2 to 1.3x10(6) CD41+ cells by day 10 when cultured in the presence of growth factor combinations 1, 2, or 3. In contrast, 2x10(5) BM CD34+ cells produced 5x10(5) CD41+ cells after 9 days in the presence of combination 1, whereas lower numbers of CD41+ cells were generated in cultures with combinations 2 and 3 (2.3x10[5] and 4.2x10[4], respectively). The addition of MGDF to cultures that were grown with combination 1 for 5 days increased the number of CD41+ cells (1.7-fold increase in PB-derived cultures, 1.6-fold increase in BM-derived cultures). Treatment with MGDF alone resulted in higher frequencies of MK progenitors than those obtained in cultures with combined growth factors (79% in PB-derived cultures, 25% in BM-derived cultures), but because total cell growth was attenuated, absolute numbers of MK progenitors were lower (7x10(5) in PB-derived cultures, 7x10(4) in BM). Morphological analysis of immunocytochemically identified megakaryocytic cells revealed mononuclear cells as the predominant cell type in all of the cultures. During the 10-day culture period, PB-derived MK progenitors did not show notable maturation, even under the influence of MGDF, whereas in BM-derived cultures MGDF induced a significant shift to binuclear cells and stage I MK after day 5. Phenotypic analysis of cell surface markers showed that the majority of cultured megakaryocytic cells coexpressed CD34 and platelet glycoproteins (GPs), also indicating an immature stage of development. The ex vivo proliferative activity of CD34+ cells and their potential to develop into the megakaryocytic lineage demonstrated considerably high interpatient variations. There was no correlation between platelet recovery following HDC with hematopoietic cell support and the magnitude of GP+ cell expansion ex vivo, suggesting the feasibilty of MK expansion ex vivo in patients with prolonged thrombocytopenia posttransplantation. In summary, these data indicate that GCSF-mobilized CD34+ PBPCs are more effectively expanded ex vivo into the megakaryocytic lineage than are CD34+ BMPCs. CD34+/GP+ MK progenitors may be an appropiate cell population for transplantion as prophylaxis or treatment of prolonged thrombocytopenia. The efficacy of this procedure will be tested prospectively in a clinical trial.

Adult↗
PubMed1996

Distinct 3p21.3 deletions in lung cancer and identification of a new human semaphorin.

Loss of chromosome 3p is a critical event in the pathogenesis of lung cancer. Overlapping homozygous 3p21.3 deletions in lung cancer cell lines involving GNAI2 were characterized and found to involve a region of genomic instability. A new widely expressed Semaphorin, H.SemaIV, was isolated from the GNAI2 deletion region. Reduced H.SemaIV expression allowed identification of additional cell lines with submicroscopic or larger deletions of the locus which occurred in a heterogeneous manner. We also demonstrate the presence of a distinct 3p21.3 homozygous deletion region, adjacent to the DNA mismatch repair gene, hMLH1, and identified deletions in direct tumors. This appears to represent one of the first demonstrations of homozygous deletions affecting 3p in direct lung tumors.

Amino Acid Sequence↗
PubMed1996

Neutral endopeptidase: variable expression in human lung, inactivation in lung cancer, and modulation of peptide-induced calcium flux.

Neutral endopeptidase (NEP; CALLA, CD10, EC 3.4.24.11) is a cell surface endopeptidase that hydrolyses bioactive peptides, including the bombesin-like peptides, as well as other neuropeptides. Bombesin-like peptides and other neuropeptides are autocrine growth factors for both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Low expression of NEP has been reported in SCLC and NSCLC cell lines. NEP inhibition has been shown to increase proliferation in one cell line. To date, NEP expression has not been quantitatively evaluated in normal adult lung, SCLC or NSCLC tumors, paired uninvolved lung from the same patient, or in other pulmonary neoplasms such as mesotheliomas and carcinoids. We examined the expression of NEP in these tissues and human cell lines using immunohistochemistry, flow cytometry, enzyme activity, ELISA, Western blot, and reverse transcription (RT)-PCR. Uninvolved lung tissue from different individuals displayed considerable variation in NEP activity and protein. By immunohistochemistry, NEP expression was detectable in alveolar and airway epithelium, fibroblasts of normal lung, and in mesotheliomas, whereas it was undetectable in most SCLC, adenocarcinoma, squamous cell carcinoma, and carcinoid tumors of the lung. NEP activity and protein levels were lower in all SCLC and adenocarcinoma tumors when compared to adjacent uninvolved lung, often at levels consistent with expression derived from contaminating stroma. NEP expression and activity were reduced or undetectable in most SCLC and lung adenocarcinoma cell lines. NEP mRNA by RT-PCR was not expressed or was in low abundance in the majority of lung cancer cell lines. The majority of lung tumors did not express NEP by RT-PCR as compared with normal adjacent lung. In addition, recombinant NEP abolished, whereas an NEP inhibitor potentiated, the calcium flux generated by neuropeptides in some lung cancer cell lines, demonstrating potential physiological significance for low NEP expression. NEP, therefore, is a signal transduction and possibly a growth modulator for both SCLC and NSCLC, emphasizing the role of neuropeptides in the pathogenesis of the major histological forms of lung cancer.

Adenocarcinoma↗
PubMed1996

Bone marrow metastases.

This article discusses the clinical significance of bone marrow metastases and the current methods being used to detect tumor cells in marrow. The strategies being investigated for eradicating cancer cells from marrow in patients receiving hematopoietic cell autografts also are reviewed.

Bone Marrow Neoplasms↗
PubMed1996

Reclassification of a polycyclic aromatic hydrocarbon-metabolizing bacterium, Beijerinckia sp. strain B1, as Sphingomonas yanoikuyae by fatty acid analysis, protein pattern analysis, DNA-DNA hybridization, and 16S ribosomal DNA sequencing.

A bacterium isolated from a polluted stream, capable of metabolizing biphenyl, naphthalene, phenanthrene, and higher-molecular-weight polycyclic aromatic hydrocarbons (D. Gibson, V. Mahadevan, D. Jerina, H. Yagi, and H. Yeh, Science 189:295-297, 1975), was previously identified as Beijerinckia sp. strain B1. In this investigation, 16S rRNA gene sequencing, biochemical tests, fatty acid methyl ester analysis, polyacrylamide gel electrophoresis of protein, and DNA-DNA hybridization were used to determine the taxonomic relationship of Beijerinckia sp. strain B1. The sequence of the 16S rRNA gene of B1 was identical to that of Sphingomonas yanoikuyae ATCC 51230T. The biochemical tests, fatty acid analysis, and sodium dodecyl sulfate-polacrylamide gel electrophoresis profile of soluble proteins of strain B1 showed results similar to those of S. yanoikuyae. DNA-DNA hybridization indicated that B1 and S. yanoikuyae ATCC 51230T are 75% homologous at the DNA level. We propose that Beijerinckia sp. strain B1 be reclassified as S. yanoikuyae.

Bacterial Proteins↗
PubMed1996

Endotoxin induces cardiac HSP70 and resistance to endotoxemic myocardial depression in rats.

Endotoxin (bacterial lipopolysaccharide, LPS) depresses myocardial function. However, heat shock and sublethal LPS can confer cardiac resistance to postischemic dysfunction. We hypothesized that a prior exposure to LPS stress induces the expression of cardiac heat shock protein 70 (HSP70) and resistance to endotoxemic myocardial depression. Moreover, induction of HSP70 by hyperthermia should also increase cardiac resistance to LPS toxicity. LPS (500 micrograms/kg ip) depressed rat left ventricular developed pressure (LVDP) maximally at 6 h (58.4 +/- 3.72 vs. 101 +/- 1.46 mmHg in saline control, P < 0.01), and myocardial contractile function recovered at 24 h. In rats pretreated with LPS 24 h earlier, subsequent LPS exposure did not depress LVDP (97.0 +/- 3.53 mmHg at 6 h, P < 0.01 vs. single exposure). Both LPS and hyperthermia (42 degrees C, 15 min) induced HSP72 mainly in the cardiac interstitial cells, including macrophages at 24 h after treatment. When hyperthermia-pretreated animals were similarly challenged with LPS, myocardial depression at 6 h was partially abrogated (LVDP 80.1 +/- 5.67 vs. 62.2 +/- 4.91 mmHg in sham+LPS group, P < 0.01). We conclude that LPS induces HSP70 in rat heart and that an exposure to LPS or heat stress confers cardiac resistance to endotoxemic myocardial depression.

Animals↗
PubMed1996

Examination of p53 alterations and cytokeratin expression in sputa collected from patients prior to histological diagnosis of squamous cell carcinoma.

Mutations in the p53 gene are detected in greater than 50% of squamous cell carcinomas of the lung and to a lesser extent in adenocarcinomas. The p53 protein is also overexpressed in a relatively high percentage of preinvasive lesions of the bronchial epithelium. However, unlike tumor tissue, immunoreactivity does not necessarily imply that cells in preinvasive lesions carry a mutant p53 allele. In some cases, overexpression may result from a cellular checkpoint reaction to a toxic or mutagenic substance such as exposure to tobacco smoke. In any case, p53 overexpression in preinvasive lesions may serve as a biomarker for high risk assessment of lung cancer and other tumors in the aerodigestive tract. A study was designed to retrospectively analyze p53 overexpression in cells from sputum samples collected prior to histological tumor diagnosis. The rationale was based on the observation that both preinvasive and tumor cells from the bronchial epithelium are exfoliated into the airways and can be detected based on morphology in sputa. Two sets of cases were chosen: 1) patients whose first primary tumor was a squamous cell carcinoma containing a mutant p53 allele with overexpression observed in most of the tumor cells; and 2) patients whose squamous cell tumor did not contain a mutant p53 allele. Cells which stained positive for p53 expression were observed in sputum samples collected from all six patients whose tumors were positive for a mutant p53 allele. Also p53 positive cells were detected on sputum slides for two of the five cases where the tumor DNA did not contain a mutation and/or tumor cells which overexpress p53 were not detected in tissue sections. Although cells which stained positive for p53 were present in sputum from patients whose tumors contained a missense mutation, the presence of p53 overexpression was not specific for tumors which contain an altered p53 allele since overexpression was detected in sputum cells from patients whose tumor DNA did not contain a p53 mutation and/or tumor cells which stained positive for p53 were not observed in tissue sections. However, the p53 positive cells in sputa collected from the latter group of patients could have been exfoliated from other lesions which contained a mutant p53 allele. The accumulation of p53 in some sputum cells was concomitant with expression of simple epithelial type cytokeratins (CK) 8 and 18 or at least one of the other cytokeratins detected by a broad spectrum (PAN) CK antibody mixture. These data imply that most of the sputum cells which overexpress p53 are epithelial cells. Moreover, our results are consistent, at least in part, with other observations that cells which overexpress p53 in dyplasias and hyperplasias express CK 8, 18. We will continue to explore the possibility that expression of cytokeratins 8, 18 and/or other cytokeratins in conjunction with p53 overexpression and/or morphological criteria could define a new class of atypical cells which are predisposed to cancer development.

Alleles↗