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Biomedical subjects

L A Chandler

Publications and source records attributed to L A Chandler.

At least 19 recordsLinked to original sources
PubMed2001

Matrix immobilization enhances the tissue repair activity of growth factor gene therapy vectors.

Although growth factor proteins display potent tissue repair activities, difficulty in sustaining localized therapeutic concentrations limits their therapeutic activity. We reasoned that enhanced histogenesis might be achieved by combining growth factor genes with biocompatible matrices capable of immobilizing vectors at delivery sites. When delivered to subcutaneously implanted sponges, a platelet-derived growth factor B-encoding adenovirus (AdPDGF-B) formulated in a collagen matrix enhanced granulation tissue deposition 3- to 4-fold (p < or = 0.0002), whereas vectors encoding fibroblast growth factor 2 or vascular endothelial growth factor promoted primarily angiogenic responses. By day 8 posttreatment of ischemic excisional wounds, collagen-formulated AdPDGF-B enhanced granulation tissue and epithelial areas up to 13- and 6-fold (p < 0.009), respectively, and wound closure up to 2-fold (p < 0.05). At longer times, complete healing without excessive scar formation was achieved. Collagen matrices were shown to retain both vector and transgene products within delivery sites, enabling the transduction and stimulation of infiltrating repair cells. Quantitative PCR and RT-PCR demonstrated both vector DNA and transgene mRNA within wound beds as late as 28 days posttreatment. By contrast, aqueous formulations allowed vector seepage from application sites, leading to PDGF-induced hyperplasia in surrounding tissues but not wound beds. Finally, repeated applications of PDGF-BB protein were required for neotissue induction approaching equivalence to a single application of collagen-immobilized AdPDGF-B, confirming the utility of this gene transfer approach. Overall, these studies demonstrate that immobilizing matrices enable the controlled delivery and activity of tissue promoting genes for the effective regeneration of injured tissues.

Adenoviridae↗
PubMed2000

Collagen-embedded platelet-derived growth factor DNA plasmid promotes wound healing in a dermal ulcer model.

BACKGROUND: Gene therapy has shown limited efficacy for treating congenital diseases, partly due to temporary gene expression and host immune responses. Such results suggest that gene therapy is ideal for chronic wound treatment where limited duration of target gene expression is required. This study tested the wound healing effects of topically applied platelet-derived growth factor (PDGF)-A or -B chain DNA plasmids embedded within a collagen lattice. MATERIALS AND METHODS: Four 6-mm dermal ulcer wounds were created in the ears of young adult New Zealand White rabbits made ischemic by division of the central and rostral arteries. Wounds were treated with lyophilized collagen containing PDGF-B DNA (1.0-3.0 mg), PDGF-A DNA (1.0 mg), irrelevant DNA (1.0 mg), or collagen alone. Wounds were dressed and harvested after 10 days for measurement of granulation tissue formation, epithelialization, and wound closure. Results were evaluated with a paired two-tailed Student t test, with P values < 0.05 considered significant. RESULTS: PDGF-B DNA increased new granulation tissue (NGT) formation up to 52% and epithelialization 34% compared with controls. Wound closure was increased up to threefold. At 1.0 mg DNA, PDGF-A and PDGF-B stimulated similar responses. No difference in NGT or epithelialization was seen between control groups. CONCLUSIONS: PDGF DNA gene therapy is effective at accelerating wound healing in ischemic dermal ulcers and provides a viable alternative to peptide growth factor therapy.

Animals↗
PubMed2000

FGF2-Targeted adenovirus encoding platelet-derived growth factor-B enhances de novo tissue formation.

Gene therapy has yet to achieve reproducible clinical efficacy, due to inadequate gene delivery, inadequate gene expression, or dose-limiting toxicity. We have developed a gene therapy technology for tissue repair and regeneration that employs a structural matrix for DNA delivery. The matrix holds the DNA vector at the treatment site and provides a scaffolding for in-growth and accumulation of repair cells and efficient DNA transfection. We now report, for the first time, matrix-mediated delivery of targeted DNA vectors for soft tissue repair. A collagen matrix was used to deliver an adenoviral vector encoding platelet-derived growth factor-B (AdPDGF-B), resulting in efficient transgene expression in vitro and in vivo. Increases in the overall levels of expression and in the relative amounts of secreted PDGF-BB were achieved when AdPDGF-B was conjugated to fibroblast growth factor (FGF2) such that the virus was targeted for cellular uptake via FGF receptors. Matrix-mediated delivery of AdPDGF-B enhanced wound healing responses in vivo, and FGF2 targeting generated effects comparable to nontargeted vectors at significantly lower doses. Therefore, matrix-mediated delivery in combination with FGF2 targeting overcomes some of the safety and efficacy limitations of current gene therapy strategies and is an attractive therapeutic approach for tissue repair and regeneration.

Animals↗
PubMed1999

Prevalent expression of fibroblast growth factor (FGF) receptors and FGF2 in human tumor cell lines.

Basic fibroblast growth factor (FGF2) has potent mitogenic and angiogenic activities that have been implicated in tumor development and malignant progression. The biological effects of FGF2 and other members of the FGF ligand family are mediated by 4 transmembrane tyrosine kinase receptors (FGFRs). To better understand the roles of FGFRs in cancer, the expression of FGF2 and each of the 4 FGFRs was assessed by RNase protection analysis of 60 human tumor cell lines, representing 9 tumor types. Expression of at least one FGFR isoform was detected in 90% and FGF2 mRNA in 35% of the cell lines. Our comprehensive analysis of FGF2 and FGFR expression in human tumor cell lines provides evidence that FGF signaling pathways are active in a majority of human tumor cell lines, and lends support to the development of anti-tumor strategies that target FGFRs.

Fibroblast Growth Factor 2↗
PubMed1998

Targeted delivery of DNA encoding cytotoxic proteins through high-affinity fibroblast growth factor receptors.

Nonviral DNA delivery strategies for gene therapy have generally been limited by a lack of specificity and efficacy. However, ligand-mediated endocytosis can specifically deliver DNA in vitro to cells bearing the appropriate cognate receptors. Similarly, in order to circumvent problems related to efficacy, DNA must encode proteins with high intrinsic activities. We show here that the ligand basic fibroblast growth factor (FGF2) can target FGF receptor-bearing cells with DNA encoding therapeutic proteins. Delivery of genes encoding saporin, a highly potent ribosomal inactivating protein, or the conditionally cytotoxic herpes simplex virus thymidine kinase, a protein that can kill cells by activating the prodrug ganciclovir, is demonstrated. The saporin gene was codon optimized for mammalian expression and demonstrated to express functional protein in a cell-free assay. FGF2-mediated delivery of saporin DNA or thymidine kinase DNA followed by ganciclovir treatment resulted in a 60 and 75% decrease in cell number, respectively. Specificity of gene delivery was demonstrated in competition assays with free FGF2 or with recombinant soluble FGF receptor. Alternatively, when histone H1, a ligand that binds to cell surface heparan sulfate proteoglycans ("low-affinity" FGF receptors), was used to deliver DNA encoding thymidine kinase, no ganciclovir sensitivity was observed. These findings establish the feasibility of using ligands such as FGF2 to specifically deliver genes encoding molecular chemotherapeutic agents to cells.

Amino Acid Sequence↗
PubMed1998

Targeting tumor cells via EGF receptors: selective toxicity of an HBEGF-toxin fusion protein.

Over-expression of the epidermal growth factor receptor (EGFR) is a hallmark of numerous solid tumors, thus providing a means of selectively targeting therapeutic agents. Heparin-binding epidermal growth factor (HBEGF) binds to EGFRs with high affinity and to heparan sulfate proteoglycans, resulting in increased mitogenic potential compared to other EGF family members. We have investigated the feasibility of using HBEGF to selectively deliver a cytotoxic protein into EGFR-expressing tumor cells. Recombinant fusion proteins consisting of mature human HBEGF fused to the plant ribosome-inactivating protein saporin (SAP) were expressed in Escherichia coli. Purified HBEGF-SAP chimeras inhibited protein synthesis in a cell-free assay and competed with EGF for binding to receptors on intact cells. A construct with a 22-amino-acid flexible linker (L22) between the HBEGF and SAP moieties exhibited an affinity for the EGFR that was comparable to that of HBEGF. The sensitivity to HBEGF-L22-SAP was determined for a variety of human tumor cell lines, including the 60 cell lines comprising the National Cancer Institute Anticancer Drug Screen. HBEGF-L22-SAP was cytotoxic in vitro to a variety of EGFR-bearing cell lines and inhibited growth of EGFR-over-expressing human breast carcinoma cells in vivo. In contrast, the fusion protein had no effect on small-cell lung carcinoma cells, which are EGFR-deficient. Our results demonstrate that fusion proteins composed of HBEGF and SAP exhibit targeting specificity and cytotoxicity that may be of therapeutic value in treating a variety of EGFR-bearing malignancies.

Epidermal Growth Factor↗
PubMed1996

Targeting DNA to cells with basic fibroblast growth factor (FGF2).

Ligand-mediated targeting of DNA was validated by condensing a plasmid DNA encoding the beta-galactosidase (beta-gal) gene with a basic fibroblast growth factor (FGF2) that was first chemically conjugated to polylysine (K). The conditions that gave optimal binding of this FGF2 to DNA also generated the highest level of beta-gal expression when added to FGF2 target cells like COS-1, 3T3, baby hamster kidney (BHK), or endothelial cells. This beta-gal activity increased in a time- and dose-dependent manner and was dependent on the inclusion of FGF2 in the complex. FGF receptor specificity was demonstrated by competition of the complex with FGF2 and heparin, and by the failure of cytochrome c or histone H1 to mimic the gene-targeting effects of FGF2. The expression of beta-gal was also endosome dependent because chloroquine increased beta-gal expression 8-fold and endosome disruptive peptides increased expression of beta-gal 26-fold. Taken together these findings establish that DNA can be introduced into cells through the high affinity FGF receptor complex, and while its efficiency will require significant enhancements to achieve sustained and elevated transgene expression, the possibility that the technique could be used to deliver DNAs encoding cytotoxic molecules is discussed.

3T3 Cells↗
PubMed1995

A nuclear post-transcriptional mechanism mediates the induction of fibronectin by glucocorticoids.

Treatment of the human fibrosarcoma cell line HT-1080 with glucocorticoids results in the induction of fibronectin (FN) protein and mRNA synthesis. We tested the contribution of transcriptional and post-transcriptional mechanisms in the regulation of FN by the synthetic glucocorticoid dexamethasone (DEX). Using nuclear run-on experiments, we found that the DEX-dependent induction of FN occurs primarily at the post-transcriptional level. The half-life of total FN mRNA was not affected by hormone treatment indicating that the induction of FN gene expression is not due to stabilization of the mature message. Interestingly, the induction by DEX was present at the level of nuclear FN RNA. We found that polyadenylation and alternative splicing of the ED-B domain of the FN transcript were not affected by glucocorticoid treatment. However, DEX was found to increase the steady-state level of unspliced FN transcript. Our data indicate that DEX exerts its effect on FN expression predominantly at the post-transcriptional level by a mechanism that, unlike most examples of post-transcriptional regulation by glucocorticoids, acts in the nucleus. Furthermore, they suggest that glucocorticoids activate a mechanism to stabilize the unspliced FN RNA. In an attempt to localize the FN RNA sequences mediating the DEX-dependent induction, we performed transfection analyses of FN minigene constructs. We suggest that the DEX-dependent regulatory elements are located in the introns since no such elements were found in the 8 kb FN mRNA.

Alternative Splicing↗
PubMed1994

A novel mechanism of Ha-ras oncogene action: regulation of fibronectin mRNA levels by a nuclear posttranscriptional event.

Although loss of cell surface fibronectin (FN) is a hallmark of many oncogenically transformed cells, the mechanisms responsible for this phenomenon remain poorly understood. The present study utilized the nontumorigenic human osteosarcoma cell line TE-85 to investigate the effects of induced Ha-ras oncogene expression on FN biosynthesis. TE-85 cells were stably transfected with metallothionein-Ha-ras fusion genes, and the effects of metal-induced ras expression on FN biosynthesis were determined. Induction of the ras oncogene, but not proto-oncogene, was accompanied by a decrease in total FN mRNA and protein levels. Transfection experiments indicated that these oncogene effects were not due to reduced FN promoter activity, suggesting that a posttranscriptional mechanism was involved. The most common mechanism of posttranscriptional regulation affects cytoplasmic mRNA stability. However, in this study the down-regulation of FN was identified as a nuclear event. A component of the ras effect was due to a mechanism affecting accumulation of processed nuclear FN RNA. Mechanisms that would generate such an effect include altered RNA processing and altered stability of the processed message in the nucleus. There was no effect of ras on FN mRNA poly(A) tail length or site of polyadenylation. There was also no evidence for altered splicing at the ED-B domain of FN mRNA. This demonstration of nuclear posttranscriptional down-regulation of FN by the Ha-ras oncogene identifies a new level at which ras oncoproteins can regulate gene expression and thus contribute to development of the malignant phenotype.

Alternative Splicing↗
PubMed1992

Coping strategies used by adolescents in dealing with family relocation overseas.

This study investigated: (a) the types of coping strategies adolescents commonly employ in response to family relocation overseas; and (b) whether sex, age, self-concept, behavioral adjustment, and previous history of moves affect the types of strategies used by these sojourners. For this purpose, we selected a widely used model of coping which distinguishes between approach and avoidance strategies and a sample of teenagers whose families had recently relocated in Belgium. Four assessment instruments were utilized: the Background Information Data Sheet, the Stress Response Scale, the Piers-Harris Children's Self-Concept Scale, and the Coping Responses Inventory--Youth Form. Results indicated that adolescent sojourners selected both approach and avoidance strategies to cope with family relocation overseas. Correlational analyses further showed that females and older teenagers tended to rely significantly more on approach strategies than on avoidance strategies. Self-concept and behavioral adjustment were also found to be related to the type coping strategies employed, while the history of previous relocation was not.

Adaptation, Psychological↗
PubMed1991

Posttranscriptional down-regulation of fibronectin in N-ras-transformed cells.

The basal level of fibronectin (FN) biosynthesis is greatly reduced in N-ras-transformed human fibrosarcoma HT1080 cells when compared with normal fibroblasts. The rate of FN biosynthesis can be significantly increased in HT1080 cells by the synthetic glucocorticoid dexamethasone, which has been shown to act by selectively stabilizing the FN message (D.C. Dean, R.F. Newby, and S. Bourgeois, J. Cell Biol., 106: 2159-2170, 1988). The present study demonstrates that the basal levels of FN biosynthesis and accumulated FN mRNA are also reduced in HT1080 cells when compared with nontumorigenic revertants of HT1080 cells in which the mutated N-ras allele is underrepresented. In contrast, the FN promoter is more active in HT1080 cells than in the revertant cells. These results indicate that the down-regulation of FN in HT1080 cells is linked to expression of the N-ras oncogene and that the effect is posttranscriptional. Therefore, the N-ras oncogene and glucocorticoids have opposite posttranscriptional effects on FN biosynthesis in HT1080 cells. However, this study demonstrates that the stimulatory effects of glucocorticoids are not mediated by a transcriptional repression of the N-ras gene.

Blotting, Northern↗
PubMed1988

Hypomethylation of DNA in the regulation of gene expression.

Although numerous possible functions of eukaryotic DNA methylation have been proposed (Ehrlich and Wang, 1981), current evidence suggests an association between hypomethylation of specific gene regions and transcriptional activity (Doerfler, 1983; Riggs and Jones, 1983). Considerable excitement has been generated by the finding that potential methylation sites are often clustered at the 5' regions of genes (Bird et al., 1985). Furthermore, these clusters appear to be protected from methylation when associated with actively expressed genes. These findings have generated a new line of thinking among researchers, namely that genes may be regulated by domains of methylation sites (Jones, 1986). Although the evidence supporting a role for methylation in eukaryotic gene regulation is convincing, it is by no means unequivocal (Bird, 1984). To date, however, determination of the methylation status of specific cytosine residues in DNA has been limited by the experimental tools at hand. Therefore, development of a method that allows for evaluation of the methylation status of all CpG sites within, and in the vicinity of, genes may be necessary to elucidate the exact relationship between DNA methylation and gene expression. The emerging consensus is that DNA methylation is one component of a hierarchy of control mechanisms that regulate eukaryotic gene expression and cellular differentiation. Furthermore, the realization that methylation plays an important role in regulating gene expression during normal cellular differentiation suggests that aberrations in this potential controlling mechanism may be implicated in the abnormal gene expression seen in cancer. Indeed, a large body of experimental evidence demonstrates altered levels and patterns of methylation in tumor cells (Riggs and Jones, 1983; Jones, 1986). The regulatory function of 5-mCyt is most likely exerted via DNA-protein interactions. Two key questions remain unanswered: How does 5-mCyt affect these interactions? More importantly, what are the proteins that modulate DNA methylation during differentiation?

5-Methylcytosine↗
PubMed1987

Allele-specific methylation of the human c-Ha-ras-1 gene.

The methylation status of individual c-Ha-ras-1 alleles in human cells was measured by Hpall/Mspl analysis of a polymorphic (VTR) region. The gene was extensively methylated in leukocytes and sperm, with the former exhibiting a highly specific methylation pattern. Individual ras alleles were differentially methylated at the VTR region in fetal fibroblasts and immortal cell lines. A new polymorphism in the 5'-flanking region of the gene was also detected. The presence or absence of an Xhol site showed a striking and complete concordance with the length of the VTR region and suggested that the site had been lost by a previous allele-specific mutation.

Alleles↗
PubMed1986

Southern blot analysis of DNA extracted from formalin-fixed pathology specimens.

We have developed a method for the extraction of DNA from formalin-fixed, paraffin-embedded pathology specimens. High-molecular-weight DNA was recovered from well-fixed nonautolyzed samples of viable tissue. DNA recovered from samples exposed to picric acid or mercuric chloride containing fixatives was not intact. Increasing the formalin fixation time decreased the amount of intact DNA available. When these limitations were taken into consideration, the procedure allowed for the removal of degraded and chemically modified DNA from the preparation, and the final product was suitable for quantitative and qualitative analysis by Southern or dot blotting techniques. Digestion with methylation-sensitive restriction endonucleases showed that DNA methylation patterns were not altered after formalin fixation.

DNA↗
PubMed1986

Patterns of DNA methylation and gene expression in human tumor cell lines.

Restriction endonuclease analysis was used to determine the methylation status of collagen, c-Ha-ras, and thymidine kinase genes in human fibroblasts and tumor cell lines. When digested with the methylation sensitive enzymes HpaII or HhaI, the DNA of each cell line generated a unique banding pattern for each gene examined. No generalized trend of gene hypomethylation or decreases in overall cytosine methylation were observed in tumor cell lines when compared to fibroblasts. Collagen biosynthetic profiles were also determined, and no correlations could be made between patterns of type I and type III collagen gene methylation and expression. Our findings support those of a previous report in which methylation and expression of the chick alpha 2(I) collagen gene were examined, and this represents the first such analysis of the pro-alpha 1 (III) collagen gene. MspI restriction fragment length polymorphisms were detected within c-Ha-ras, pro-alpha 2(I) collagen, pro-alpha 1(III) collagen, and thymidine kinase genes. The ras gene polymorphisms can be attributed to variation in the number of tandem repeats within a MspI fragment at the 3' end of the gene. The other gene polymorphisms may be due to base pair mutations at methylated cytosine residues within CCGG sequences.

Cell Line↗
PubMed1985

The incidence of stressful life events of elementary school-aged children.

Certain events, commonly found in the life histories of children, have been characterized as potentially stressful (Rutter, 1981). There is some evidence to support the hypothesis that these events play a role in the etiology of various disorders, although the nature of that relationship is not clear (Dohrenwend & Dohrenwend, 1974). Yet that relationship remains the subject of considerable interest, as can be seen by the number of studies on the influence of stressful life events on children's emotional adjustment and school performance (Boike, Gesten, Cowen, Felner, & Francis, 1978; Felner, Ginter, Boike, & Cowen, 1981; Felner, Stolberg, & Cowen, 1975; Gesten, Scher, & Cowen, 1981; Felner, Stolberg, & Cowen, 1975; Gesten, Scher, & Cowen, 1978; Rolf & Garmezy, 1974; Sandler & Block, 1979; Siegel, 1981). Sandler and Ramsey (1980) have suggested that the role of life events as stressors can be understood to be a function of, among other things, the frequency of occurrence of events. For research and clinical assessment purposes, baseline data on the incidence of stressful life events of children seem to be necessary. In order to collect and organize baseline data, the relevant demographic variables must be identified. It was hypothesized that there might be two relevant demographic variables: age and socioeconomic status (SES). The purposes of this study were to (a) conduct a survey to gather baseline data on the incidence of stressful life events of children, and (b) examine the effects of age and SES on the number of events reported.

Child↗
PubMed1983

Empirical classification of emotional adjustment reactions.

The validity of a diagnostic classification scheme for the emotional adjustment reactions of children is tested, and empirical findings are offered in support of a descriptive approach to classification. Results validate clinical observations of four types of children commonly seen for emotional adjustment reactions. Further research is proposed to develop instruments for personality assessment that would be sensitive to these categories.

Adjustment Disorders↗