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Biomedical subjects

J M Ford

Publications and source records attributed to J M Ford.

At least 37 records · Page 2Linked to original sources
PubMed2000

Xeroderma pigmentosum p48 gene enhances global genomic repair and suppresses UV-induced mutagenesis.

UV-damaged DNA-binding activity (UV-DDB) is deficient in some xeroderma pigmentosum group E individuals due to mutation of the p48 gene, but its role in DNA repair has been obscure. We found that UV-DDB is also deficient in cell lines and primary tissues from rodents. Transfection of p48 conferred UV-DDB to hamster cells, and enhanced removal of cyclobutane pyrimidine dimers (CPDs) from genomic DNA and from the nontranscribed strand of an expressed gene. Expression of p48 suppressed UV-induced mutations arising from the nontranscribed strand, but had no effect on cellular UV sensitivity. These results define the role of p48 in DNA repair, demonstrate the importance of CPDs in mutagenesis, and suggest how rodent models can be improved to better reflect cancer susceptibility in humans.

Animals↗
PubMed2000

p53-mediated DNA repair responses to UV radiation: studies of mouse cells lacking p53, p21, and/or gadd45 genes.

Human cells lacking functional p53 exhibit a partial deficiency in nucleotide excision repair (NER), the pathway for repair of UV-induced DNA damage. The global genomic repair (GGR) subpathway of NER, but not transcription-coupled repair (TCR), is mainly affected by p53 loss or inactivation. We have utilized mouse embryo fibroblasts (MEFs) lacking p53 genes or downstream effector genes of the p53 pathway, gadd45 (Gadd45a) or p21 (Cdkn1a), as well as MEFs lacking both gadd45 and p21 genes to address the potential contribution of these downstream effectors to p53-associated DNA repair. Loss of p53 or gadd45 had a pronounced effect on GGR, while p21 loss had only a marginal effect, determined by measurements of repair synthesis (unscheduled DNA synthesis), by immunoassays to detect removal of UV photoproducts from genomic DNA, and by assays determining strand-specific removal of CPDs from the mouse dhfr gene. Taken together, the evidence suggests a role for Gadd45, but relatively little role for p21, in DNA repair responses to UV radiation. Recent evidence suggests that Gadd45 binds to UV-damaged chromatin and may affect lesion accessibility. MEFs lacking p53 or gadd45 genes exhibited decreased colony-forming ability after UV radiation and cisplatin compared to wild-type MEFs, indicating their sensitivity to DNA damage. We provide evidence that Gadd45 affects chromatin remodelling of templates concurrent with DNA repair, thus indicating that Gadd45 may participate in the coupling between chromatin assembly and DNA repair.

Animals↗
PubMed2000

Proapoptotic p53-interacting protein 53BP2 is induced by UV irradiation but suppressed by p53.

p53 is an important mediator of the cellular stress response with roles in cell cycle control, DNA repair, and apoptosis. 53BP2, a p53-interacting protein, enhances p53 transactivation, impedes cell cycle progression, and promotes apoptosis through unknown mechanisms. We now demonstrate that endogenous 53BP2 levels increase following UV irradiation induced DNA damage in a p53-independent manner. In contrast, we found that the presence of a wild-type (but not mutant) p53 gene suppressed 53BP2 steady-state levels in cell lines with defined p53 genotypes. Likewise, expression of a tetracycline-regulated wild-type p53 cDNA in p53-null fibroblasts caused a reduction in 53BP2 protein levels. However, 53BP2 levels were not reduced if the tetracycline-regulated p53 cDNA was expressed after UV damage in these cells. This suggests that UV damage activates cellular factors that can relieve the p53-mediated suppression of 53BP2 protein. To address the physiologic significance of 53BP2 induction, we utilized stable cell lines with a ponasterone A-regulated 53BP2 cDNA. Conditional expression of 53BP2 cDNA lowered the apoptotic threshold and decreased clonogenic survival following UV irradiation. Conversely, attenuation of endogenous 53BP2 induction with an antisense oligonucleotide resulted in enhanced clonogenic survival following UV irradiation. These results demonstrate that 53BP2 is a DNA damage-inducible protein that promotes DNA damage-induced apoptosis. Furthermore, 53BP2 expression is highly regulated and involves both p53-dependent and p53-independent mechanisms. Our data provide new insight into 53BP2 function and open new avenues for investigation into the cellular response to genotoxic stress.

Apoptosis↗
PubMed2000

Reduced global genomic repair of ultraviolet light-induced cyclobutane pyrimidine dimers in simian virus 40-transformed human cells.

The p53 tumor-suppressor gene has been implicated in the inducible activation of excision repair of ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPDs) in human cells. Because the large T antigen (LTAg) of the simian virus 40 (SV40) binds p53 protein and can interfere with its function, it was of interest to study DNA repair in normal human fibroblasts that had been transformed by SV40 compared with that in their nontransformed parental counterparts and to determine whether such transformation attenuated global genomic repair (GGR) of CPDs. Three methods were used to measure GGR in UV-irradiated cells: (i) an immunoassay using monoclonal antibodies specific for CPDs or 6-4 photoproducts (6-4PPs), (ii) zone sedimentation in alkaline sucrose gradients to measure the average DNA strand size after specific nicking at CPD sites in duplex DNA with T4 endonuclease V (TEV), and (iii) Southern hybridization of TEV-treated DNA with strand-specific mRNA probes to assess removal of CPDs from either strand of a defined genetic sequence in an expressed gene. Whereas repair of 6-4PPs was very similar in paired SV40-transformed and primary fibroblasts, GGR of CPDs was significantly reduced in the SV40-transformed cells. In contrast, SV40 transformation did not appreciably affect the efficiency of transcription-coupled repair. These data support the hypothesis that SV40 transformation can result in reduced levels of GGR, most likely because of the inhibition of normal p53 function by LTAg.

Cell Line, Transformed↗
PubMed1999

P300 amplitude is related to clinical state in severely and moderately ill patients with schizophrenia.

BACKGROUND: Relationships between illness severity and neurobiologic abnormalities in schizophrenia were studied in subpopulations varying in clinical severity. METHODS: Auditory ERPs were collected from 28 severely ill, chronically hospitalized schizophrenic men from a state hospital; 29 moderately ill inpatient and outpatient schizophrenic men from a veterans hospital; and 30 healthy male subjects from the community as controls. Clinical symptoms were evaluated in patients using the Brief Psychiatric Rating Scale (BPRS). RESULTS: Both schizophrenic patient groups had smaller P300 amplitude than the control subjects. Severely ill patients had smaller P300s than moderately ill patients and scored higher on three BPRS factor scores as well as BPRS Total. Among severely ill patients, P300 amplitude was unrelated to clinical symptoms. Among moderately ill patients, P300 was related to Withdrawal/Retardation, Anxiety/Depression, and BPRS Total. After combining patients, Thinking Disturbance emerged as an additional correlate of P300. Group differences in P300 could not be accounted for by group differences in symptom severity using analysis of covariance. CONCLUSIONS: Reduced P300 amplitude marks the diagnosis of schizophrenia, but also reflects individual differences in severity, including positive symptoms. Previous failures to find relationships between positive symptoms and P300 may have been due to a restricted range of clinical severity.

Acute Disease↗
PubMed1999

Failures of automatic and strategic processing in schizophrenia: comparisons of event-related brain potential and startle blink modification.

Noises elicit startle blinks that are inhibited when immediately (approximately 100 ms) preceded by non-startling prepulses, perhaps reflecting automatic sensory gating. Startle blinks are facilitated when preceded by prepulses at longer lead intervals, perhaps reflecting strategic processes. Event-related brain potentials (ERPs) and startle blinks were used to investigate the well-documented prepulse inhibition failure in schizophrenia. Blinks and ERPs were recorded from 15 schizophrenic men and 20 age-matched controls to noises alone and to noises preceded by prepulses at 120 (PP120), 500 (PP500) and 4000 ms (PP4000) lead intervals. Neither blinks nor any of the ERP components elicited by the noise alone differentiated schizophrenics from controls, although responses to noises were modified by prepulses differently in the two groups. With the N1 component of the ERP, patients showed normal inhibition but lacked facilitation, and with P2, patients lacked inhibition, but showed normal facilitation. With reflex blinks and P300, inhibition was seen in both groups, but no facilitation. These results suggest that different neural circuits are involved in blink and cortical reflections of startle modification in schizophrenics and controls, with both automatic and strategic processes being impaired in schizophrenia.

Acoustic Stimulation↗
PubMed1999

Expression of the p48 xeroderma pigmentosum gene is p53-dependent and is involved in global genomic repair.

In human cells, efficient global genomic repair of DNA damage induced by ultraviolet radiation requires the p53 tumor suppressor, but the mechanism has been unclear. The p48 gene is required for expression of an ultraviolet radiation-damaged DNA binding activity and is disrupted by mutations in the subset of xeroderma pigmentosum group E cells that lack this activity. Here, we show that p48 mRNA levels strongly depend on basal p53 expression and increase further after DNA damage in a p53-dependent manner. Furthermore, like p53(-/-) cells, xeroderma pigmentosum group E cells are deficient in global genomic repair. These results identify p48 as the link between p53 and the nucleotide excision repair apparatus.

DNA Damage↗
PubMed1999

Schizophrenia: the broken P300 and beyond.

Many of the social, economic, and political problems facing people with schizophrenia are due to a misconception in the community that schizophrenia is not a biologically based disease but a myth. Because the diagnosis is based on self-reported symptoms, it is difficult for many people to acknowledge that schizophrenia is real. One goal of psychophysiological research has been to anchor both diagnosis and symptoms in biological reality. Reduction of the amplitude of the P300 component of the event-related potential (ERP) is the most replicable biological marker of the disease. Data are presented suggesting that P300 is both a state and a trait marker of the disease and may be sensitive to the progressive/degenerative course of the disease. Although the P300 tracks changes in clinical symptoms, it remains reduced even in patients in relative remission. P300 amplitude reduction is related to enduring negative symptoms, waning of attention, and gray matter volume deficits. ERP components other than P300 can also manifest the biological reality of various symptoms of the disease.

Brain↗
PubMed1998

Hepatitis B x protein inhibits p53-dependent DNA repair in primary mouse hepatocytes.

The mechanisms by which the hepatitis B x protein (HBx) contributes to hepatocarcinogenesis remain unclear. However, interaction with the tumor suppressor gene p53 and inhibition of p53-dependent cellular functions, including nucleotide excision repair, could be central to this process. We studied the levels of global repair (removal of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts) and transcription-coupled repair (removal of CPDs in both strands of the dihydrofolate reductase gene) in primary wild-type and p53-null mouse hepatocytes. We show that global repair of CPDs appears to be more efficient in mouse hepatocytes than in other commonly studied rodent cells and approaches the levels of human cells and that p53 is required for global genomic DNA repair of CPDs but not for transcription-coupled repair. We then investigated the effect of HBx expression on hepatocyte nucleotide excision repair. We demonstrate that HBx expression affects DNA repair in a p53-dependent manner. Transient HBx expression reduces global DNA repair in wild-type cells to the level of p53-null hepatocytes and has no effect on the repair of a transfected damaged plasmid. Therefore, in viral hepatitis, the hepatitis B virus could inhibit the p53-dependent component of global repair leading, over time, to accumulation of genetic defects and fostering carcinogenesis.

Animals↗
PubMed1998

Human fibroblasts expressing the human papillomavirus E6 gene are deficient in global genomic nucleotide excision repair and sensitive to ultraviolet irradiation.

We investigated the role of wild-type p53 activity in modulating nucleotide excision repair after UV irradiation in normal and p53-deficient primary human fibroblasts created by expression of the human papillomavirus 16 E6 gene. Compared with parental cells, the E6-expressing fibroblasts were deficient in global genomic repair of both UV-induced cyclobutane pyrimidine dimers and 6-4 photoproducts but exhibited normal transcription-coupled repair. The E6-expressing cells were also more sensitive than their parental counterparts to UV irradiation and displayed similar levels of UV-induced apoptosis. These results suggest that disruption of wild-type p53 function by E6 expression results in selective loss of p53-dependent global genomic nucleotide excision repair, but not UV-induced apoptosis, leading to enhanced UV sensitivity.

Apoptosis↗
PubMed1997

Expression of wild-type p53 is required for efficient global genomic nucleotide excision repair in UV-irradiated human fibroblasts.

We have shown previously that Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in the removal of UV-induced cyclobutane pyrimidine dimers from genomic DNA, but still proficient in the transcription-coupled repair pathway (Ford, J. M., and Hanawalt, P. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8876-8880). We have now utilized monoclonal antibodies specific for cyclobutane pyrimidine dimers or 6-4 photoproducts, respectively, to measure their repair in UV-irradiated human fibroblasts. Cells homozygous for p53 mutations were deficient in the repair of both photoproducts, whereas cells heterozygous for mutant p53 exhibited normal repair of 6-4 photoproducts, but decreased initial rates of removal of cyclobutane pyrimidine dimers, compared with normal cells. The specificity of the effect of wild-type p53 on nucleotide excision repair was demonstrated in a p53 homozygous mutant cell line containing a tetracycline-regulated wild-type p53 gene. Wild-type p53 expression and activity were suppressed in the presence of tetracycline, whereas withdrawal of tetracycline resulted in the induction of p53 expression, cell cycle checkpoint activation, and DNA damage-induced apoptosis. The regulated expression of wild-type p53 resulted in the recovery of normal levels of repair of both cyclobutane pyrimidine dimers and 6-4 photoproducts in genomic DNA, but did not alter the transcription-coupled repair of cyclobutane pyrimidine dimers. Therefore, the wild-type p53 gene product is an important determinant of nucleotide excision repair activity in human cells.

Cells, Cultured↗
PubMed1997

Combined event-related fMRI and EEG evidence for temporal-parietal cortex activation during target detection.

Target detection is the process of bringing a salient stimulus into conscious awareness. Target detection evokes a prominent event-related potential (ERP) component (P3) in the electroencephalogram (EEG). We combined the high spatial resolution of functional magnetic resonance imaging (fMRI) with the high temporal resolution of EEG to investigate the neural generators of the P3. Event-related brain activation (ERBA) and ERPs were computed by time-locked averaging of fMRI and EEG, respectively, recorded using the same paradigm in the same subjects. Target detection elicited significantly greater ERBAs bilaterally in the temporal-parietal cortex, thalamus and anterior cingulate. Spatio-temporal modelling of ERPs based on dipole locations derived from the ERBAs indicated that bilateral sources in the temporal-parietal cortex are the main generators of the P3. The findings provide convergent fMRI and EEG evidence for significant activation of the temporal-parietal cortex 285-610 ms after stimulus onset during target detection. The methods developed here provide a novel multimodal neuroimaging technique to investigate the spatio-temporal aspects of processes underlying brain function.

Adult↗
PubMed1997

A short fractionation radiotherapy treatment for poor prognosis patients with high grade glioma.

Thirty-two patients prospectively identified as having poor prognosis high grade glioma, with a MRC prognostic score >25, were treated with a short palliative course of radiotherapy. A total dose of 36 Gy in 12 fractions was given to the tumour, including oedema and a 2 cm margin, using parallel pair fields prescribed to the midplane with MV photons. Twenty-eight patients completed treatment as planned, while four failed to complete treatment because of clinical deterioration or death. The median survival for the whole group was 16 weeks, with seven patients surviving for more than 6 months. Approximately two-thirds of the surviving patients remained at home after the completion of treatment. A matched case-control comparison with data from patients in previous MRC studies who had received a 6-week course of treatment shows that, for this group of patients, survival is similar (hazard ratio 1.0; 95% confidence interval (CI) 0.57-1.74). The 95% CI for the difference in median survival time excludes a reduction of more than 7 weeks with the 36 Gy course. This shortened radiotherapy regimen may therefore be satisfactory for most poor prognosis patients. However, patients with performance status 3 gained little benefit from treatment, and it is suggested that this group should have a trial period of assessment at home prior to a decision on treatment.

Adult↗
PubMed1996

The effect of stimulus-response incompatibility on P3 latency depends on the task but not on age.

To help identify the loci of age-related slowing of cognitive processing, the effects of stimulus-response (S-R) incompatibility and stimulus degradation on P3 latency and reaction time (RT) were assessed in ten young and ten elderly women. Subjects saw the words right, left, RIGHT, or LEFT in tasks which required responses to (1) the meaning of the word (WORD) (2) its case (CASE) and (3) both (CASE/WORD). Each task was tested with regular and degraded stimuli. On half the trials, the stimulus and response were incompatible. Although RTs and P3s of elderly subjects were slower, especially RTs in CASE/WORD, stimulus degradation and S-R incompatibility did not differentially affect the two groups, suggesting that cognitive processing of older subjects is not especially prolonged in perceptual and response-related stages. For both groups RTs and P3s were task dependent and were prolonged by degradation and S-R incompatibility. Incompatibility delayed RTs in WORD and CASE/WORD but P3s in WORD only. Thus, young and elderly subjects show qualitatively similar psychophysiological and behavioral indicators of processing speed.

Adult↗
PubMed1996

P-glycoprotein-mediated multidrug resistance: experimental and clinical strategies for its reversal.

The study of the cellular, biochemical, and molecular biology and pharmacology of MDR has provided one of the most active and exciting areas within cancer research and one that holds great promise for translation into clinical benefit. While convincing evidence for the functional role of P-gp in mediating clinical drug resistance in humans remains elusive, studies of the clinical expression of P-gp and trials of chemosensitizers with cancer chemotherapy suggest "resistance modification" strategies may be effective in some tumors with intrinsic or acquired drug resistance. However, even if P-gp-associated MDR proves to be a relevant and reversible cause of clinical drug resistance, numerous problems remain to be solved before effective clinical chemosensitization may be achieved. Such factors as absorption, distribution, and metabolism; the effect of chemosensitizers on chemotherapeutic drug clearance; toxicity to normal tissues expressing P-gp; and the most efficacious modulator regimens all remain to be defined in vivo. Clearly, the identification of more specific, potent, and less clinically toxic chemosensitizers for clinical use remains critical to the possible success of this approach. Nonetheless, the finding that a number of pharmacological agents can antagonize a well-characterized form of experimental drug resistance provides promise for potential clinical applications. Further study of chemosensitizers in humans and the rational design of novel chemosensitizers with improved activity should define the importance of MDR in clinically resistant cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗