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Biomedical subjects

J C Lang

Publications and source records attributed to J C Lang.

At least 19 recordsLinked to original sources
PubMed2000

Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Adenocarcinoma↗
PubMed1999

p16 mutation frequency and clinical correlation in head and neck cancer.

The tumor suppressor gene p16, when altered, has been shown to play a role in oncogenesis in many different tumor types including head and neck cancer. The goal of this study was to analyse alterations to p16 in squamous cell carcinoma (SCC) of the head and neck and to correlate these with clinical outcome. RNA was isolated from 26 SCC head and neck tumors and from 24 matched controls. A reverse transcription polymerase chain reaction was utilized to generate p16 cDNA, which was sequenced and analysed for alterations. In the 26 patient group 58% of the tumors had a p16 alteration, which were characterized by: 8 deletions, 1 insertion/deletion, 4 point mutations and 2 with no p16 expression. In 24 matched normal tissue samples there were no p16 alterations. Those patients with p16 alterations appear to have survival rates comparable to those without p16 alterations, although patients with p16 alterations appear to have more recurrences.

Carcinoma, Squamous Cell↗
PubMed1998

Frequent mutation of p16 in squamous cell carcinoma of the head and neck.

RNA was isolated from 22 squamous cell carcinomas (SCCs) obtained from diverse sites within the head and neck and from matched normal tissue where available. Tissue samples were then screened for expression of RNA from tumor suppressor gene p16 by utilizing semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis. p16-Specific PCR amplification products generated from tumor samples were subject to further analysis by direct DNA sequencing to determine if any tumor sample harbored a p16 mutation. The results show the presence of mutations in 10 of 22 (45%) of the tumor samples. Mutations comprise two identical point mutations, two small deletions (1 bp and 2 bp), one single-nucleotide insertion, four larger deletions, and an insertion/deletion. No mutations in p16 have been identified by analysis of PCR products generated from normal matched tissue, suggesting that p16 alterations are generated by somatic mutation and are not germline in origin. All 22 samples were analyzed additionally by immunohistochemistry for nuclear expression of the retinoblastoma (RB) tumor suppressor gene product. Results show lack of RB nuclear expression in only one sample, suggesting that mutation of RB is an infrequent event in the development of SCC of the head and neck (SCCHN).

Carcinoma, Squamous Cell↗
PubMed1997

GRS, a novel member of the Bcl-2 gene family, is highly expressed in multiple cancer cell lines and in normal leukocytes.

Our laboratory previously described the independent isolation of the fibroblast growth factor 4 (FGF-4) gene by NIH3T3 transformation assay using DNA from a patient with CML leukemia (Lucas et al., 1994). The FGF-4 gene was truncated by DNA rearrangement with a novel gene named GRS. In this manuscript we describe isolation of GRS cDNA and show by sequence comparison that GRS is a novel member of the Bcl-2 gene family. Northern analysis shows expression of the gene in normal human tissue to be largely restricted to the hematopoietic compartment. Analysis of the pattern of gene expression in cancer cell lines demonstrates GRS is expressed in hematopoietic malignancies and in melanoma. The chromosomal location of GRS has also been determined. The gene is positioned on chromosome 15 within bands q24-25.

Base Sequence↗
PubMed1997

Activation of fibroblast growth factor 8 gene expression in human embryonal carcinoma cells.

To study the role of fibroblast growth factor 8 (FGF-8) in human development and malignancies, we have isolated and characterized its gene. The gene spans 6.0 kbp and is comprised of five exons. Using reverse transcription-polymerase chain reaction, we were able to show that FGF-8 is expressed in two of the seven human mammary carcinoma cell lines tested and in only one of nine breast tumors. In contrast, both of the two normal breast tissues tested express FGF-8. FGF-8 was previously shown to be present in adult testis and ovary. Surprisingly, only one of the seven testis carcinomas and one of 12 ovary carcinomas express FGF-8, whereas all three kidney carcinomas tested express FGF-8. We further showed that fetal brain and lung express FGF-8, whereas fetal intestine and liver do not. Finally, we showed that a teratocarcinoma cell line, Tera-2, can be induced to express FGF-8 mRNA by fetal bovine serum.

Adult↗
PubMed1997

Mutation and downregulation of the transforming growth factor beta type II receptor gene in primary squamous cell carcinomas of the head and neck.

In the present study, we analyzed 28 squamous cell carcinomas of the head and neck (SCCHN) for mutations in the coding region of TbetaR-II using 'Cold' SSCP and automatic DNA sequencing analyses. Twenty-one percent (6/28) of the SCCHN examined contained TbetaR-II mutations compared with patient-matched normal tissues. These alterations included five missense mutations (A:T-->G:C transitions in codons 250, 401, 448 and 488, and a G:C-->T:A transversion in codon 373), and a 38-bp deletion between nucleotides 1825 to 1862. In addition to these code-altering mutations, one case exhibited a silent mutation (A:T-->G:C transition in codon 451) and three cases contained one of two potential population polymorphisms (codons 354 and 389). In contrast to colon and gastric cancers exhibiting microsatellite instability (MI) or replication errors (RER+), no 'indirect' frameshift mutations were identified within a 10-bp polyadenine repeat present in the TbetaR-II coding sequence. All of the mutations in the present study occurred within the highly conserved serine/threonine kinase domain and represent the first report of such 'direct' TbetaR-II mutations in primary human tumors. In addition, we analyzed a subset of SCCHN and corresponding normal samples for TbetaR-II mRNA expression using semi-quantitative multiplex RT-PCR. Expression of TbetaR-II was decreased by 24% to 74% in 20 of 23 SCCHN (87%) compared with patient-matched normal tissues. Taken together, the results from this study suggest that alterations in the nucleic acid sequence and mRNA expression of TbetaR-II are prevalent events in the development of SCCHN, which may deregulate cell cycle control.

Carcinoma, Squamous Cell↗
PubMed1995

Regulation of FGF-4 enhancer activity by transcription factor NF-Y.

Regulation of FGF-4 gene expression is controlled both by elements in the promoter and by an enhancer domain located in the untranslated region of the third exon. We have determined that transcription factor NF-Y binds to the FGF-4 promoter. We further show by mutational analysis that binding of NF-Y is essential for FGF-4 enhancer activity but has minimal effect on activity of the FGF-4 promoter alone.

Animals↗
PubMed1995

Increased in vitro and in vivo tumoricidal activity of a macrophage cell line genetically engineered to express IFN-gamma, IL-4, IL-6, or TNF-alpha.

Genetically engineered monocytes and macrophages may have potential as effector cells for the adoptive immunotherapy of cancer. As a first step, we have transfected the genes encoding either mouse interferon (IFN)-gamma, human interleukin (IL)-6, mouse IL-4, or mouse tumor necrosis factor (TNF)-alpha into the mouse macrophage cell line, J774A.1 cells using retroviral vectors. In vitro activation of J774A.1 cells by gene modification was assessed by morphological changes, proliferative activity was determined by [3H]-TdR uptake, and cytolytic activity was assessed using an 18-hour chromium-51 (51Cr) release assay. In vivo tumoricidal activity was studied by means of local adoptive immunotherapy using intratumoral injection of transfected effector cells. IFN-gamma gene-transfected J774A.1 [J7(IFN-gamma)] cells developed filamentous processes, increased doubling times, and enhanced tumoricidal activity against three tumor cell lines: the TNF-sensitive fibrosarcoma line WEHI 164 and the TNF-alpha-resistant cell lines B16 melanoma and C1300 neuroblastoma. IL-6-, TNF-alpha-, and IL-4-gene-transfected J774A.1 cells also had augmented tumoricidal activity but did not display any changes in morphology or growth. Cytolytic activity was markedly reduced after the addition of anti-TNF-alpha antibodies. Cytolytic J7(IFN-gamma) cells showed upregulated expression of TNF-alpha messenger RNA. After intratumoral injection of J7(IL-4) and J7(IFN-gamma) cell mixtures, 50% of established B16 melanomas were rejected by C57BL/6 mice, thereby demonstrating synergistic killing. Further studies on gene-transfected macrophages should better define their potential usefulness in tumor immunotherapy.

3T3 Cells↗
PubMed1994

The FGF-4 promoter is required for transformation and is active in both embryonal and somatic cells.

We report the independent isolation of a rearranged FGF-4 gene from a patient with chronic myeloid leukaemia. We show that the FGF-4 gene has been truncated 30 nucleotides 3' to the coding sequence and has been fused to the RNA processing signals from a putative unknown gene on chromosome 15. We demonstrate that the promoter region of the FGF-4 gene is active in NIH3T3 cells and is indeed necessary for transformation. Using the luciferase reporter assay we have shown that the FGF-4 5' flanking sequences possess easily detectable promoter activity in both F9 and HeLa cell lines. 5' deletion analysis of the FGF-4 promoter has delineated regions containing cis-acting elements of functional importance. These regulatory regions are common to both embryonal and somatic cell lines. Electrophoretic mobility shift assay, using nuclear extracts from F9 and HeLa cells, has allowed detection of DNA-protein interactions occurring in the functionally significant regions. Subsequent comparison of the human and murine FGF-4 promoters show that the regions of functional significance are highly conserved. We suggest that the FGF-4 gene may be suppressed through a distal suppressor locus and becomes active when separated from this suppressor.

3T3 Cells↗
PubMed1993

Changes in in vivo protein-DNA interactions occur at the c-myc P2 promoter during differentiation.

Down regulation of the c-myc gene is a prerequisite for the differentiation of a number of cell types. Studies have shown that two mechanisms of inactivation are involved in c-myc repression: a block of the elongation of RNA polymerase followed by transcriptional inactivation mediated through promoter sequences. In this study DMS in vivo footprinting was performed on the P2 promoter region of c-myc in differentiated and undifferentiated HL60 cells. A differentiation-specific footprint was observed at G residues immediately upstream of the TATA box. This observation occurred only in cells differentiated for 48 hours or more and hence is likely to be involved in the repression of c-myc by promoter inactivation.

Base Sequence↗
PubMed1993

c-MYC repression of promoter activity through core promoter elements.

c-MYC was found to repress expression of the c-myc promoter in a transient assay system. Deletion constructs showed the cis-acting element(s) were common to both the P1 and P2 promoters. Further analysis involving replacement of the P2 TATA box and initiator elements with the SV40 late promoter initiator localized the cis-acting elements to the core promoter.

Dose-Response Relationship, Drug↗
PubMed1992

Drug release profiles of ophthalmic formulations. 1. Instrumentation.

An experimental method is described for measuring time-release profiles of drugs from various ophthalmic dosage forms. This in vitro method is carried out under conditions nearly representative of those observed in vivo. Specifically, we have reproduced in vitro the small volume and the slow exchange rate of the human precorneal tear reservoir. Volumes on the order of 8-30 microL are achievable. The concentration of drug downstream from the sample reservoir is analyzed nearly continuously and the entire release profiles are appropriate for modeling. This analytical system has been used to study drug release kinetics from controlled release formulations such as gels and suspensions. The method is compared with alternative techniques.

Calibration↗
PubMed1992

The metabolism of 14C-tazadolene succinate in the dog.

1. Beagle dogs dosed orally with 14C-tazadolene succinate excreted much of the dose in the urine (mean 63.1% in 5 days with most excreted in the first 24 h). A lesser proportion of the dose was excreted in the faeces (mean 20.7%) and again most of this was voided in the first 24 h. 2. Four metabolites were identified and quantified in the urine, namely 3-hydroxy-(M1), 4-hydroxy- (M2a), and 3-methoxy-4-hydroxy-tazadolene (M2b) and N-[2-(phenylmethylene)cyclohexyl]-beta-alanine (M3). 3. In the 24 h urine, M2a and b glucuronides accounted for 17.7% dose, unconjugated M2a and b for 11.3%, and M3 for 18.3%. Insufficient M1 was present to be quantified. The same metabolites were seen in the 24 h faeces, but at lower concn. Thus M2a and b glucuronides, M2a and b, and M3 were 3.2%, 4.9% and 3.5% dose respectively. 4. All three phenols were present in plasma as their glucuronides as well as the beta-alanine derivative. They all had the same tmax of 2 h and t1/2 lambda 1 of the order of 1 h.

Analgesics↗
PubMed1992

Measurement of oxybutynin and its N-desethyl metabolite in plasma, and its application to pharmacokinetic studies in young, elderly and frail elderly volunteers.

1. A quantitative h.p.l.c. plasma assay for oxybutynin (OB) and its active metabolite, N-desethyl oxybutynin (DEOB) is described. The method is linear with coefficients of variation ranging between 4 and 11.8% for OB and 4.6-9.1% for DEOB over the typical concentration range measured. Minimum detectable levels were 0.5 and 5 ng/ml for OB and DEOB respectively from a 2 ml sample. 2. Pharmacokinetic parameters were obtained after a single oral dose of OB and after administration two or three times daily to frail elderly and elderly volunteer groups respectively. Single dose results were also compared with data from young healthy volunteers. 3. There was a wide range in peak blood levels and high levels of parent drug were matched by high DEOB metabolite levels. Plasma levels on repeated administration were as would be predicted from the single dose kinetics. 4. Area under the plasma time course curve for DEOB metabolite was less than or equal to 5 than that of the parent drug. 5. A trend of increasing peak plasma levels and bioavailability was observed with increasing age and frailty, with the differences more apparent between the active elderly and frail elderly groups than between the active elderly and young volunteers. 6. Results indicate that for frail elderly patients a lower initial starting dose of 2.5 mg OB given two or three times a day may provide adequate therapeutic blood levels of the drug.

Administration, Oral↗
PubMed1991

Limits on optimizing ocular drug delivery.

The problem of optimizing ocular bioavailability of topically applied ophthalmic drugs is discussed. A formula for drug concentration in the tear film is derived using well-known pharmacokinetic relationships and a first-order drug decay model for the tear film. The time integral of the tear film concentration is then related to ocular bioavailability. The results of this analysis show that: (1) high corneal permeability (corresponding to lipophilic compounds) produces the highest bioavailability; (2) the bioavailability of drugs with high corneal permeability is relatively unaffected by drug volume; and (3) by making the dosage volume sufficiently small, a bioavailability improvement factor of approximately 4 can be obtained for drugs with low corneal permeability.

Administration, Topical↗
PubMed1991

Regulation of AP-1/DNA complex formation in vitro.

The level of AP-1 DNA-binding activity exhibited in vitro by unfractionated extracts of Hela nuclei can be stimulated by a low molecular weight fraction from rabbit reticulocyte lysate. Stimulation also requires a heat labile component of the nuclear extract, probably a protein. Stimulated and unstimulated extracts with high and low AP-1 DNA-binding activities contain the same levels of proteins reactive with antisera against Jun and Fos, proteins which are shown to be involved in the AP-1/DNA complexes detected in vitro. The low molecular weight fraction from reticulocyte lysate can be substituted by the reducing agent dithiothreitol (DTT) in the stimulation reaction and conversely oxidised glutathione greatly reduces formation of AP-1/DNA complexes. The binding activities of transcription factors SP-1, NF-1 and CBP are unaffected by DTT or oxidised glutathione. These observations, taken together, suggest that the efficiency with which pre-existing Fos and Jun proteins can bind an AP-1 target sequence in vitro can be controlled by a nuclear activity which is sensitive to oxidation/reduction and that this control mechanism is specific for AP-1.

Animals↗