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Biomedical subjects

J Kirk

Publications and source records attributed to J Kirk.

At least 91 records · Page 5Linked to original sources
PubMed1994

Selective toxicity of TGF-alpha-PE40 to EGFR-positive cell lines: selective protection of low EGFR-expressing cell lines by EGF.

The sensitivity of human breast and lung cancer cell lines to TGF-alpha-PE40, a novel chimeric recombinant cytotoxin composed of two independent domains, (i) TGF-alpha and (ii) a 40 kDa segment of the Pseudomonas exotoxin protein, PE-40, was investigated. Toxicity varied widely, correlated with epidermal growth factor receptor (EGFR) levels (P = 0.01) and was greatly reduced by EGF, indicating that binding of TGF-alpha-PE40 to EGFR is important in mediating toxicity. Cell lines expressing low EGFR levels were most highly protected by EGF, indicating that normal (low EGFR-expressing) tissue may be selectively protected by EGF in vivo. P-glycoprotein did not confer resistance to TGF-alpha-PE40, and toxicity was unaffected by multidrug resistance-modulating agents (cyclosporin A, tamoxifen, verapamil), indicating a role for TGF-alpha-PE40 in the clinical management of drug-resistant tumours.

Breast Neoplasms↗
PubMed1994

HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney.

Studies assessing mechanisms of proximal tubular cell (PTC) physiology and pathophysiology increasingly utilize cell culture systems to avoid the complexity of whole organ/whole animal experiments. However, no well-differentiated PTC line derived from adult human kidney currently exists. Therefore, the goal of this research was to establish such a line by transduction with human papilloma virus (HPV 16) E6/E7 genes. A primary PTC culture from normal adult human renal cortex was exposed to a recombinant retrovirus containing the HPV 16 E6/E7 genes, resulting in a cell line designated HK-2 (human kidney-2) which has grown continuously in serum free media for more than one year. HK-2 cell growth is epidermal growth factor dependent and the cells retain a phenotype indicative of well-differentiated PTCs (positive for alkaline phosphatase, gamma glutamyltranspeptidase, leucine aminopeptidase, acid phosphatase, cytokeratin, alpha 3 beta 1 integrin, fibronectin; negative for factor VIII-related antigen, 6.19 antigen and CALLA endopeptidase). Furthermore, HK-2 cells retain functional characteristics of proximal tubular epithelium (Na+ dependent/phlorizin sensitive sugar transport; adenylate cyclase responsiveness to parathyroid, but not to antidiuretic, hormone). The E6/E7 genes are present in the HK-2 genome, as determined by PCR. To assess its potential usefulness as a tool for studying injury and repair, HK-2 cells were exposed to a toxic concentration of H2O2 +/- iron chelation (deferoxamine) or hydroxyl radical scavenger (Na benzoate) therapy. Only the former blocked H2O2 cytotoxicity, reproducing results previously obtained with freshly isolated rat proximal tubular segments. In conclusion, an immortalized adult human PTC line has been established by transduction with HPV 16 E6/E7 genes. It appears to be well-differentiated on the basis of its histochemical, immune cytochemical, and functional characteristics, and it can reproduce experimental results obtained with freshly isolated PTCs. Thus, this new PTC line could have substantial research application.

Adult↗
PubMed1994

Rapid RT-PCR amplification from limited cell numbers.

We describe a rapid and efficient RT-PCR method particularly suited to procedures involving limited cell and target gene copy numbers. Purified leukocytes and myeloid colonies derived from patients with chronic myelogenous leukemia (CML) in chronic phase were used for direct RT-PCR. Purified cells and colonies were lysed using a small quantity of DEPC-treated water containing RNasin as an RNA inhibitor. The untreated lysate was either used immediately for RT-PCR or frozen at -70 degrees C for later use. By this method we were able to consistently amplify bcr-abl transcripts from as few as 10 cells. No noticeable difference was observed between products amplified from fresh and frozen samples.

Base Sequence↗
PubMed1994

Association of measles virus with neurofibrillary tangles in subacute sclerosing panencephalitis: a combined in situ hybridization and immunocytochemical investigation.

Neurofibrillary tangle formation, a cardinal characteristic of Alzheimer's disease, is also a feature of several other neurodegenerative disorders, including subacute sclerosing panencephalitis (SSPE). In the present study the association of measles virus genome with neurofibrillary tangle formation has been studied in five cases of SSPE, using in situ hybridization (measles genome) and immunocytochemistry (tau, ubiquitin and beta/A4 amyloid). In two cases with duration of disease less than one year, neurofibrillary tangle formation was not observed. However, in those cases in which the disease was of several years duration, numerous tau- and ubiquitin-positive neurofibrillary tangles were demonstrated. In the two cases of longest duration, double-labelling techniques demonstrated the frequent association of neurofibrillary tangle formation with neuronal measles virus genome positivity. Immunocytochemistry for beta/A4 amyloid failed to demonstrate amyloid in any of the five cases. These findings support the hypothesis that neurofibrillary tangle formation can occur independently of amyloid formation and that this mechanism may operate in both Alzheimer's disease and in virally-induced disease.

Adolescent↗
PubMed1994

Detection of BCR-ABL fusion genes in adult acute lymphoblastic leukemia by the polymerase chain reaction.

The sensitivity and clinical utility of the polymerase chain reaction (PCR) assay for the detection of BCR-ABL gene rearrangement was compared to conventional cytogenetics for the Philadelphia chromosome (Ph1) in adult acute lymphoblastic leukemia (ALL) patients entered onto a single clinical trial. Ninety-three patients had evaluable PCR assays for both the p190bcr-abl and p210bcr-abl type of BCR-ABL gene rearrangements. Twenty-one of 93 patients (23%) were positive for the BCR-ABL rearrangement by the PCR assay. Fourteen of these patients had the p210brc-abl BCR-ABL rearrangement characteristically seen in CML patients, while seven had the p190bcr-abl rearrangement seen in ALL alone. Of 61 patients analyzed, both with conventional cytogenetics and PCR, eight (13%) were positive for the Ph1, while 14 (23%) were positive for the BCR-ABL rearrangement by the PCR assay. Discordance between the PCR assay and cytogenetics occurred in eight cases where the PCR assay was positive and the cytogenetics negative, and two cases where the PCR assay was negative and cytogenetics positive. PCR positivity did not correlate with treatment response, survival, or relapse-free survival, but there was a higher percentage of L2 FAB morphology in the PCR+ cases compared to the PCR-cases (67 vs. 28%, p = 0.003). In addition, the data suggested that patients with a p190bcr-abl rearrangement have a better response to induction therapy, but a worse relapse-free survival compared to patients with a p210bcr-abl breakpoint, but these differences were not statistically significant. These data suggest that PCR and conventional cytogenetics may provide complementary information, since there appear to be a subset of patients who are Ph1-negative yet BCR-ABL positive by PCR. Further studies will be required to determine the prognostic significance of the detailed information about BCR-ABL breakpoints that is available from the PCR assay.

Adolescent↗
PubMed1993

Volume-regulatory taurine release from a human lung cancer cell line. Evidence for amino acid transport via a volume-activated chloride channel.

Exposure of a human lung epithelial cancer cell-line to hypo-osmotic media led to a marked increase in the rate of efflux from the cells of taurine, a non-essential sulfonic amino acid. The osmotically-activated taurine efflux was inhibited by a range of known Cl- channel blockers, the most potent of which were NPPB and 1,9-dideoxyforskolin. These reagents were similarly effective at inhibiting the osmotically-activated efflux of I-, a known substrate of volume-activated Cl- channels. The results are consistent with the hypothesis that volume-regulatory taurine release from these cells is mediated by a volume-activated Cl- channel.

Amino Acids↗
PubMed1993

The effect of hypoxia on acquired drug resistance and response to epidermal growth factor in Chinese hamster lung fibroblasts and human breast-cancer cells in vitro.

Prolonged hypoxia induced transient drug resistance in Chinese hamster lung fibroblasts. Previously hypoxic cells were resistant to adriamycin and resistant to etoposide. Complete recovery of etoposide sensitivity was observed following reaeration for 24 hr. A change in P-glycoprotein expression was unlikely to contribute to the resistance caused by hypoxia, since adriamycin resistance was not reversed by verapamil. However, alteration in the plasma membrane structure may be involved, since previously hypoxic cells were resistant to extracellular superoxide radical generated by the addition of xanthine/xanthine oxidase. In contrast, adriamycin sensitivity was not altered by hypoxia in 3 human breast-cancer cell lines. MDA-468 and MCF-7/Adr differed in their response to EGF, independent of the presence of hypoxia. These results suggest that hypoxic-stress-induced drug resistance is not generalized.

Animals↗
PubMed1993

Measles virus infection of cells in perivascular infiltrates in the brain in subacute sclerosing panencephalitis: confirmation by non-radioactive in situ hybridization, immunocytochemistry and electron microscopy.

As part of continuing multidisciplinary studies on the neuropathogenesis of subacute sclerosing panencephalitis (SSPE), in situ hybridisation, immunocytochemistry and electron microscopy were used to detect measles virus nucleic acid, protein and nucleocapsids in brain perivascular infiltrates of three cases. Perivascular cuffing cells which contained measles virus nucleic acid and antigens were found in all cases. Infected cuffs occurred predominantly in areas of general parenchymal cell infection and in many of these a high proportion of the infiltrating cells were infected. Other cuffs in these areas were either uninfected or contained only a few infected cells. Occasional infected cells were also seen in cuffs in non-infected areas. In contrast, no specific immunocytochemical reactions or in situ hybridisation for measles virus was observed in brain tissue from a patient with herpes encephalitis. By electron microscopy viral nucleocapsid, consistent with measles virus, was found within the cytoplasm of plasma cells in the inflammatory cuffs in SSPE brain tissue. Possible explanations for our results are that infiltrates become infected on arrival in the CNS or alternatively, that the infected infiltrates reflect a generalised infection of the reticuloendothelial system. The frequent presence of uninfected cuffs favours the former explanation.

Brain↗
PubMed1993

Selective reversal of vinblastine resistance in multidrug-resistant cell lines by tamoxifen, toremifene and their metabolites.

In this study we describe the effects of tamoxifen, toremifene and their 4-hydroxy and N-desmethyl metabolites on the toxicity of a range of drugs to human breast and lung cancer and to Chinese hamster ovary cell lines, determined using a tetrazolium-based semi-automated colorimetric assay. Vinblastine resistance was completely abolished in an mdr1-transfected lung cancer cell line (S1/1.1), indicating that P-glycoprotein-mediated multidrug resistance can be fully reversed by anti-oestrogens. A substantial (14- to 39-fold) enhancement of vinblastine toxicity to highly multidrug-resistant (MCF-7Adr) cells expressing P-glycoprotein was also observed in the presence of tamoxifen, toremifene and their metabolites, while m-amsacrine, cisplatin and melphalan toxicity was unaffected.

Amsacrine↗
PubMed1993

Differential modulation of doxorubicin toxicity to multidrug and intrinsically drug resistant cell lines by anti-oestrogens and their major metabolites.

The ability of the anti-oestrogens tamoxifen, toremifene and their 4-hydroxy and N-desmethyl metabolites to modify doxorubicin (dox) toxicity to intrinsically resistant and multidrug resistant cell lines was compared, using human breast and lung cancer, and Chinese hamster ovary cell lines. The anti-oestrogens significantly enhanced dox toxicity to multidrug resistant, P-glycoprotein-positive cell lines, but did not affect toxicity to intrinsically resistant, P-glycoprotein-negative cells. Modification was observed at clinically achievable anti-oestrogen concentrations. Toremifene and tamoxifen would therefore appear to be good candidates for in vivo studies as MDR modulating agents in selected patients with P-glycoprotein-positive tumours.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Deletion analysis maps ocular albinism proximal to the steroid sulphatase locus.

We describe a pedigree in which four male members are affected by a contiguous gene abnormality involving the short arm of the X chromosome (Xp22.32). Bivariate flow cytometry of lymphoblastoid cell lines from two of these individuals and a normal male showed a 6-7 megabase deletion in affected males, and high resolution chromosomal G-banding of an obligate heterozygote showed the deletion to reside in the Xp22.32 region. Affected members had X-linked ichthyosis due to steroid sulphatase deficiency, Kallmann's syndrome, but no ocular albinism. In two out of four affected individuals studied, there was unilateral renal agenesis. Deletion analysis using the Xp22.32 markers MIC2, DXS31, DXS 89, GMGX9, DXS278, DXS143, and DXS9 showed that the deletion extended from DXS31 to DXS143 (inclusive). The absence of ocular albinism in this pedigree shows conclusively that the X-linked ocular albinism gene resides proximal to the DXS143 locus. Further, the inconstant association of unilateral renal agenesis with X-linked Kallmann's syndrome, even when the latter is caused by a complete deletion of the gene, suggests that the absence of the X-linked Kallmann gene can be compensated in renal development.

Albinism, Ocular↗
PubMed1993

Xp22.3 deletions in isolated familial Kallmann's syndrome.

Several familial cases of Kallmann's syndrome (KS) have been reported, among which the X-chromosome-linked mode of inheritance is the most frequent. The gene responsible for the X-linked KS has been localized to the terminal part of the X-chromosome short arm (Xp22.3 region), immediately proximal to the steroid sulfatase gene responsible for X-linked ichthyosis. Large deletions of this region have been previously shown in patients affected with both X-linked ichthyosis and KS. We report here the search for Xp22.3 deletions in 20 unrelated males affected with isolated X-linked KS. Only 2 deletions were found using Southern blot analysis, indicating that large deletions are uncommon in patients affected with KS alone. Both deletions were shown to include the entire KAL gene responsible for X-linked KS. The patients carrying these deletions exhibit additional clinical anomalies, which are discussed: unilateral renal aplasia, unilateral absence of vas deferens, mirror movements, and sensory neural hearing loss.

Adolescent↗
PubMed1993

Specific inhibitors distinguish the chloride channel and drug transporter functions associated with the human multidrug resistance P-glycoprotein.

Expression of the human multidrug resistance P-glycoprotein is associated with two activities, active drug transport and a volume-regulated chloride channel. In this study we define four classes of compound, based on their differential effects on these two activities. Class I compounds are substrates transported by P-glycoprotein. They also prevent channel activation when added to the cytoplasmic face of the membrane. Class II compounds include reversers of multidrug resistance such as verapamil. These compounds inhibit drug transport and block the chloride channel when added to the outer face of the membrane. Class III compounds include conventional channel blockers which block the chloride channel but do not influence drug transport. Class IV compounds, for example cyclosporin A, appear to inhibit drug transport but do not affect chloride channel activity. These findings have implications for the relationship between the channel and transporter functions associated with P-glycoprotein expression, and for the development of clinical agents which reverse multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Use of CT scans for the investigation of headache: a report from ASPN, Part 1.

BACKGROUND: Clinicians in the Ambulatory Sentinel Practice Network (ASPN) order computed tomography (CT) scans for approximately 3% of patients with headache. This study was undertaken to provide information about the reasons for ordering CT scans and the results obtained. METHODS: Weekly return cards were used to collect data on every patient for whom a CT scan was ordered to investigate a headache during a 19-month period. Copies of CT reports were reviewed, and a chart audit was performed to collect further clinical information whenever an intracranial tumor, subarachnoid hemorrhage (SAH), or subdural hematoma (SDH) was reported. RESULTS: Clinicians in 58 practices ordered 349 CT scans. Only 52 patients (15%) had abnormalities noted on neurological examination. Most CT scans were ordered because the clinician believed that a tumor (49%) or an SAH (9%) might be present. Fifty-nine (17%) were ordered because of patient expectation or medicolegal concerns. Of the 293 reports reviewed, 14 indicated that a tumor, an SAH, or an SDH was present. Two of the 14 (14%) were false positives. Forty-four (15%) of the reports noted incidental findings of questionable significance. CONCLUSIONS: Because there are no clear guidelines for the use of CT for the investigation of headache, physicians must exercise good clinical judgment in their attempts to identify treatable disease in a cost-effective manner. ASPN clinicians made selective use of CT scans based on a combination of factors that included physician and patient concerns. CT was an imperfect tool in this setting. Most of the positive results represented false positives or incidental findings that could have led to adverse effects and additional costs.

Adolescent↗