PubMed Health⌕ Search

Biomedical subjects

J Lunec

Publications and source records attributed to J Lunec.

At least 37 records · Page 2Linked to original sources
PubMed2001

p53 cellular localization and function in neuroblastoma: evidence for defective G(1) arrest despite WAF1 induction in MYCN-amplified cells.

This study investigated the hypothesis that p53 accumulation in neuroblastoma, in the absence of mutation, is associated with functional inactivation, which interferes with downstream mediators of p53 function. To test this hypothesis, p53 expression, location, and functional integrity was examined in neuroblastoma by irradiating 6 neuroblastoma cell lines and studying the effects on p53 transcriptional function, cell cycle arrest, and induction of apoptosis, together with the transcriptional function of p53 after irradiation in three ex vivo primary, untreated neuroblastoma tumors. p53 sequencing showed five neuroblastoma cell lines, two of which were MYCN-amplified, and that all of the tumors were wild-type for p53. p53 was found to be predominantly nuclear before and after irradiation and to up-regulate the p53 responsive genes WAF1 and MDM2 in wild-type p53 cell lines and a poorly-differentiated neuroblastoma, but not a differentiating neuroblastoma or the ganglioneuroblastoma part of a nodular ganglioneuroblastoma in short term culture. This suggests intact p53 transcriptional activity in proliferating neuroblastoma. Irradiation of wild-type p53 neuroblastoma cell lines led to G(1) cell cycle arrest in cell lines without MYCN amplification, but not in those with MYCN amplification, despite induction of WAF1. This suggests MYCN amplification may alter downstream mediators of p53 function in neuroblastoma.

Apoptosis↗
PubMed2001

Induction and excretion of ultraviolet-induced 8-oxo-2'-deoxyguanosine and thymine dimers in vivo: implications for PUVA.

Molecular epidemiology has linked ultraviolet-induced DNA damage with mutagenesis and skin carcinogenesis. Ultraviolet radiation may damage DNA in one of two ways: either directly, leading to lesions such as cyclobutane thymine dimers (T<>T), or indirectly, via photosensitizers that generate free radical species that may ultimately produce such oxidative lesions as 8-oxo-2'-deoxyguanosine. We report the results of a pilot, case control study in which seven, healthy, human volunteers (skin type II; aged 23-56 y; three male, four female) received a suberythemal dose of whole body irradiation from ultraviolet-A-emitting fluorescent tubes used in psoralen plus ultraviolet A therapy. First void, mid-stream urine samples were collected pre-exposure and daily postexposure, for up to 13 d. Analysis of urinary 8-oxo-2'-deoxyguanosine and cyclobutane thymine dimers was by competitive enzyme-linked immunosorbent assay (interassay coefficient of variation < or = 10%) and compared with a matched, control group of unirradiated individuals. A maximal increase in levels of urinary 8-oxo-2'-deoxyguanosine was seen 4 d post-ultraviolet exposure. A subsequent reduction was noted, before finally returning to baseline. Similarly, cyclobutane thymine dimer levels peaked 3 d postexposure, before returning to baseline. In contrast to the 8-oxo-2'-deoxyguanosine analysis, however, a second peak was noted at days 9-11, before again returning to baseline. This is the first report examining urinary 8-oxo-2'-deoxyguanosine and cyclobutane thymine dimers following ultraviolet exposure of healthy human subjects. This work illustrates the induction and time course for excretion of ultraviolet-induced lesions, perhaps alluding to repair and ultimately offering the potential to define psoralen plus ultraviolet A dosage regimes in terms of minimizing DNA damage and hence cancer risk.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2001

Cyclin D1 expression in transitional cell carcinoma of the bladder: correlation with p53, waf1, pRb and Ki67.

Normal cell proliferation is closely regulated by proteins called cyclins. One of these, cyclin D1, in combination with its corresponding cyclin-dependent kinase (cdk), is essential for G(1)/S phase transition. Cyclin/cdk complexes are generally inhibited by cyclin-dependent kinase inhibitors(ckis), some of which are induced by wild-type p53. The aims of this study were: to investigate levels of cyclin D1 expression in transitional cell carcinoma (TCC) of the bladder; to correlate these results with data concerning the expression of p53, waf1, pRb and Ki67; and to determine whether cyclin D1 expression could predict clinical outcome. Paraffin-sections from 150 newly diagnosed bladder tumours (Ta/T1 = 97; T2-T4 = 53) were stained for cyclin D1 using immunohistochemistry and a cyclin D1 index assigned. These results were correlated with data relating to the expression of p53 and waf1 by the same tumours. A representative subset of 54 tumours (Ta/T1 = 28; T2-T4 = 26) was also stained for Ki67 and 55 were stained for pRb. The clinical course of each patient was recorded and multivariate analyses of risk factors for tumour recurrence, stage progression and overall survival were performed. Positive staining for cyclin D1 was found in 83% of tumours. The staining pattern varied between tumours with nuclear, cytoplasmic or a combination of the two evident in different tumours. 89% of Ta/T1 and 74% of T2-T4 tumours showed nuclear staining with or without cytoplasmic staining. The median value for cyclin D1 staining was significantly higher in Ta/T1 tumours (41%) compared with T2-T4 tumours (8%, P< 0.005) with 26% of muscle-invasive tumours demonstrating absent staining. In addition, the median value for cyclin D1 staining was significantly higher in G1/G2 tumours (43%) compared with G3 tumours (14%, P< 0.005). There was a significant positive correlation between expression of cyclin D1 and waf1 expression (P< 0.0001) as well as pRb expression but not between cyclin D1 expression and expression of p53. Ki67 expression was significantly associated with increasing tumour stage (P< 0.005) and histological grade (P< 0.05) but did not correlate with cyclin D1 expression. A cyclin D1 index > or = 8% was associated with significantly better survival in those patients with muscle-invasive disease (T2-T4). In addition, there was a significantly higher progression rate for those patients with Ta/T1 disease whose tumours demonstrated cytoplasmic cyclin D1 staining. These results indicate that cyclin D1 expression is significantly higher in low-stage, well differentiated bladder tumours and strongly correlates with waf1 expression. In a multivariate analysis, cyclin D1 expression is an independent prognostic indicator of survival in those patients with muscle-invasive disease.

Adult↗
PubMed2001

The impact of p53 status on cellular sensitivity to antifolate drugs.

The impact of p53 status on cellular sensitivity to antifolate drugs has been examined in seven human cell lines (A549, MCF7, T-47D, CCRF-CEM, COR-L23, A2780, and HCT-116) and p53 nonfunctional counterparts of two of the cell lines (HCT-116/N7 and A2780/CP70). p53 status was determined by sequencing and functional assays. The sensitivities of the cell lines to growth inhibition (sulphorhodamine B assay) produced by four antifolate drugs (Alimta, methotrexate, raltitrexed, and lometrexol) were studied. There was no clear relationship between functional p53 status and sensitivity to methotrexate or lometrexol, whereas a functional p53 status was possibly associated with resistance to Alimta- and raltitrexed-induced growth inhibition. In contrast, in the two pairs of related human tumor cell lines (HCT-116 and HCT-116/N7 and A2780 and A2780/CP70) cells with functional p53 were more sensitive to Alimta- and raltitrexed-induced growth inhibition (P = 0.002). Detailed studies were performed with the A2780 cell lines, and in the parental cells sensitivity to Alimta- and raltitrexed-induced cytotoxicity (clonogenic assay) was similar to the sensitivity determined in the sulphorhodamine B assay. However, in A2780/CP70 cells, 1 microM of drug resulted in only 40-60% growth inhibition yet > or = 85% cytotoxicity. After Alimta and raltitrexed exposure for < or = 72 h, there were no differences between the A2780 and A278/CP70 cell lines in cell cycle phase distribution, absolute cell number, or the induction of apoptosis. However, the cellular protein content of the A2780/CP70 cells was 3-6-fold higher than in A2780 cells after Alimta and raltitrexed treatment, suggesting that cells without functional p53 can maintain protein synthesis in the absence of cell division (unbalanced cell growth). In conclusion, the apparent impact of functional p53 status on sensitivity to antifolate drugs may depend upon the phenotypic/genotypic background as well as the assay used to measure cellular sensitivity.

Apoptosis↗
PubMed2001

Prognostic significance of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in voided urine samples from patients with transitional cell carcinoma of the bladder.

PURPOSE: To study the role of urinary matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in bladder cancer and their relationship to tumor progression. EXPERIMENTAL DESIGN: MMP-1 and TIMP-1 were measured by ELISA in urine samples from 131 patients with bladder tumors (7 cis, 74 Ta, 29 T1, and 21 T2-T4; 46 G(1), 41 G(2), and 37 G(3)), 5 patients with prostate cancer, 33 patients with benign lower urinary tract disorders, and 36 healthy volunteers. Complete clinical data were available for 100 patients with bladder cancer with a median follow-up time of 24 months (range: 4-39 months). RESULTS: MMP-1 was detected in urine samples from 21 of 131 (16%) patients with bladder cancer but was undetectable in samples from all other groups (P < 0.0001). Urinary MMP-1 was detected in a higher percentage of patients with T2-T4 tumors and G(3) tumors than patients with cis/Ta/T1 or G(1)-G(2) tumors (P = 0.04 and P = 0.0074, respectively). Patients with detectable concentrations of urinary MMP-1 had higher rates of disease progression (P = 0.04) and death from bladder cancer (P = 0.02) than patients with undetectable urinary MMP-1. All patient groups had higher urinary TIMP-1 concentrations than healthy volunteers (P = 0.02). Patients with muscle-invasive tumors had higher concentrations of urinary TIMP-1 than patients with cis/Ta/T1 tumors (P = 0.037), but there was no association between TIMP-1 and tumor grade. Urinary TIMP-1 levels strongly correlated with tumor size (P = 0.0002). Progression-free survival rates were lower for patients with urinary TIMP-1 concentrations above the median (1.8 ng/ml, P = 0.04), but urinary TIMP-1 levels were not related to disease-specific survival. Patients with T2-T4 tumors and G(3) tumors had significantly lower urinary MMP-1:TIMP-1 ratios than patients with Ta/T1 bladder tumors (P = 0.039) or G(1)-G(2) tumors (P = 0.0415). CONCLUSIONS: Where urinary MMP-1 is detectable, the patient is more likely to have a bladder tumor of advanced stage or grade and may be at increased risk of disease progression and death of bladder cancer. The relationship between urinary TIMP-1, muscle-invasion, and disease progression in bladder cancer is at variance with its role as an inhibitor of MMPs and warrants additional evaluation.

Adult↗
PubMed2000

Simultaneous measurement of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine by HPLC-MS/MS.

An assay with high selectivity and sensitivity has been developed which, for the first time, allows quantitative, simultaneous measurement in DNA of both 8-oxo-2'-deoxyguanosine (8-oxodG) and 8-oxo-2'-deoxyadenosine (8-oxodA)-important biomarkers of oxidative DNA damage in vivo. Using reversed-phase HPLC coupled to electrospray tandem mass spectrometry (HPLC-MS/MS) in multiple reaction monitoring (MRM) mode it was possible to detect background levels of these lesions in commercially available calf thymus DNA (85 +/- 3 and 7.1 +/- 0.2 per 10(6) DNA bases for 8-oxodG and 8-oxodA respectively; n = 3). Levels of 8-oxodG determined by HPLC coupled to an electrochemical detection system (HPLC-EC) were found to be similar (75 +/- 6 per 10(6) DNA bases; n = 3) to those obtained using tandem mass spectrometry.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Aberrant processing of oxidative DNA damage in systemic lupus erythematosus.

Defective DNA damage processing has been reported in systemic lupus erythematosus (SLE). Vitamin C may modulate formation/removal of the oxidative DNA lesion 8-oxo-2'-deoxyguanosine (8-oxodG). Baseline levels of 8-oxodG measured in SLE serum, urine and PBMC DNA did not differ significantly from healthy subjects. In contrast to healthy subjects, no significant decrease in PBMC 8-oxodG or increase in urinary 8-oxodG was noted in vitamin C supplemented SLE patients. A significant, although attenuated, increase in serum 8-oxodG was detected in SLE patients, compared to healthy subjects. These data support putative abnormalities in the repair/processing of 8-oxodG in SLE.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

The effects of vitamin C supplementation on protein oxidation in healthy volunteers.

We have investigated vitamin C supplementation effects on immunoglobulin oxidation (carbonyls) and total plasma protein sulfhydryls in healthy human volunteers. After receiving placebo, plasma ascorbate and oxidation markers were unchanged. Following 5 weeks supplementation with vitamin C (400 mg/day), plasma ascorbate increased but no significant effect on protein oxidation was observed. At 10 and 15 weeks supplementation, carbonyl levels were significantly reduced (P < 0.01) in subjects with low baseline ascorbate (29.51 +/- 5.3 microM) but not in those with normal baseline ascorbate (51.81 +/- 2.3 microM). To eliminate any effect from seasonal variation in dietary antioxidant intake, a second phase was undertaken. Subjects on vitamin C for 15 weeks were randomly assigned to receive either placebo or vitamin C. No difference in plasma sulfhydryl content was observed. Subjects withdrawn from supplementation showed an increase in immunoglobulin carbonyl content (P < 0.01). This demonstrates that dietary vitamin C supplementation can reduce certain types of oxidative protein damage in subjects with low basal antioxidant.

Adult↗
PubMed2000

Expression of cyclin-dependent kinase inhibitor p21(WAF1) and p53 tumour suppressor gene in laryngeal cancer.

The prognostic value of the expression of the cyclin-dependent kinase inhibitor p21 and the p53 tumour suppressor gene was examined using immunohistochemistry in 60 patients with laryngeal cancer. Multivariate analysis using Cox's proportional hazard method, showed that p21 expression (P = 0.02) and advanced T stage (P = 0.003) significantly predicted survival. It was concluded that p21 expression may be a useful prognostic indicator in laryngeal cancer.

Aged↗
PubMed2000

Fluorescence in situ hybridization techniques for the rapid detection of genetic prognostic factors in neuroblastoma. United Kingdom Children's Cancer Study Group.

Neuroblastoma is the commonest extracranial solid tumour in children. There are a number of molecular genetic features known which are of prognostic importance and which are used to direct therapy. Identification and targeting of high-risk individuals with intensive therapeutic regimens may allow an improvement in survival rates. The most powerful biological parameters associated with prognosis in this malignancy are chromosomal changes, especially MYCN amplification, deletion of chromosome 1p and aneuploidy. Rapid characterization of these aberrations at the time of diagnosis is paramount if stratification according to risk group is to be achieved. This paper describes the rapid detection of del(1p), MYCN amplification and trisomy using interphase fluorescence in situ hybridization on imprints from fresh tumour biopsies. The results are related to those obtained by standard molecular methods and karyotyping.

Aneuploidy↗
PubMed2000

Urinary 8-oxo-2'-deoxyguanosine--source, significance and supplements.

Oxidative damage to cellular biomolecules, in particular DNA, has been proposed to play an important role in a number of pathological conditions, including carcinogenesis. A much studied consequence of oxygen-centred radical damage to DNA is 8-oxo-2'-deoxyguanosine (8-oxodG). Using numerous techniques, this lesion has been quantified in various biological matrices, most notably DNA and urine. Until recently, it was understood that urinary 8-oxodG derives solely from DNA repair, although the processes which may yield the modified deoxynucleoside have never been thoroughly discussed. This review suggests that nucleotide excision repair and the action of a specific endonuclease may, in addition to the nucleotide pool, contribute significantly to levels of 8-oxodG in the urine. On this basis, urinary 8-oxodG represents an important biomarker of generalised, cellular oxidative stress. Current data from antioxidant supplementation trials are examined and the potential for such compounds to modulate DNA repair is considered. It is stressed that further work is required to link DNA, serum and urinary levels of 8-oxodG such that the kinetics of formation and clearance may be elucidated, facilitating greater understanding of the role played by oxidative stress in disease.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Immunochemical quantitation of UV-induced oxidative and dimeric DNA damage to human keratinocytes.

There is growing evidence to suggest that solar radiation-induced, oxidative DNA damage may play an important role in skin carcinogenesis. Numerous methods have been developed to sensitively quantitate 8-oxo-2'deoxyguanosine (8-oxodG), a recognised biomarker of oxidative DNA damage. Immunoassays may represent a means by which the limitations of many techniques, principally derived from DNA extraction and sample workup, may be overcome. We report the evaluation of probes to thymine dimers and oxidative damage in UV-irradiated cells and the DNA derived therefrom. Thymine dimers were most readily recognised, irrespective of whether in situ in cells or in extracted DNA. However, using antibody-based detection the more subtle oxidative modifications required extraction and, in the case of 8-oxodG, denaturation of the DNA prior to successful recognition. In contrast, a recently described novel probe for 8-oxodG detection showed strong recognition in cells, although appearing unsuitable for use with extracted DNA. The probes were subsequently applied to examine the relative induction of lesions in cells following UV irradiation. Guanine-glyoxal lesions predominated over thymine dimers subsequent to UVB irradiation, whereas whilst oxidative lesions increased significantly following UVA irradiation, no induction of thymine dimers was seen. These data support the emerging importance of oxidative DNA damage in UV-induced carcinogenesis.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Rapid lupus autoantigen relocalization and reactive oxygen species accumulation following ultraviolet irradiation of human keratinocytes.

OBJECTIVE: In vitro treatment with ultraviolet B (UVB) induces relocalization of lupus autoantigens to the cell surface. We have addressed the relationship between autoantigen relocalization, accumulation of intracellular reactive oxygen species (ROS) and the induction of apoptosis following UVA and UVB exposure. METHODS: Human primary keratinocytes were exposed in vitro to doses of UVA and UVB equivalent to 0.01-4 times the minimal erythemal dose. The cellular locations of Ro60, Ro52, Sm, U2-B" and La were determined using monoclonal antibodies. ROS accumulation and apoptosis induction were assessed using the intracellular ROS probe 2'7'-dichlorodihydrofluorescein diacetate, and the viability stains Hoechst 33342 and propidium iodide. RESULTS: UV treatment induced the relocalization of all five autoantigens investigated and an accumulation of ROS. UVA and UVB induced necrosis and apoptosis, respectively. CONCLUSION: These data suggest that both UVA and UVB induce ROS within keratinocytes but have significantly different effects upon autoantigen relocalization and cell viability.

Antigens, Surface↗
PubMed2000

Development of a quality control material for the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an in vivo marker of oxidative stress, and comparison of results from different laboratories.

The measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine is an increasingly popular marker of in vivo oxidative damage to DNA. A random-sequence 21-mer oligonucleotide 5'-TCA GXC GTA CGT GAT CTC AGT-3' in which X was 8-oxo-guanine (8-oxo-G) was purified and accurate determination of the oxidised base was confirmed by a 32P-end labelling strategy. The lyophilised material was analysed for its absolute content of 8-oxo-dG by several major laboratories in Europe and one in Japan. Most laboratories using HPLC-ECD underestimated, while GC-MS-SIM overestimated the level of the lesion. HPLC-ECD measured the target value with greatest accuracy. The results also suggest that none of the procedures can accurately quantitate levels of 1 in 10(6) 8-oxo-(d)G in DNA.

Biomarkers↗
PubMed1999

Urinary thymine dimers and 8-oxo-2'-deoxyguanosine in psoriasis.

Psoralen in conjunction with UVA (PUVA) is perhaps the most effective treatment for psoriasis. It is, however, a risk factor for skin cancer in these patients and there is a need to develop non-invasive assays reflective of treatment-induced DNA damage. We report here the assessment of two important lesions, thymine dimer (T<>T) and 8-oxo-2'-deoxyguanosine (8-OHdG), in the urine of psoriasis patients. It was found that, once corrected for urine concentration, the psoriatic group had significantly higher (P<0. 0001) urinary levels of thymine dimers compared to the control group. No significant differences in urinary 8-OHdG levels were noted between the psoriatic, atopic dermatitis and control groups. Therefore biomonitoring of therapy from the very start with this simple and non-invasive assay could perhaps be an effective measure of the risk involved with the treatment allowing optimization for minimal-risk therapy.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1999

Discrepancies in the measurement of UVC-induced 8-oxo-2'-deoxyguanosine: implications for the analysis of oxidative DNA damage.

Ultraviolet (UV) light-induced indirect, oxidative damage to DNA has received increasing attention with respect to the mutagenic and carcinogenic effects of solar radiation. An oxidative lesion that has raised particular interest because of its qualitative and quantitative importance is 8-oxo-2'-deoxyguanosine. This deoxynucleoside lesion is most frequently measured by high performance liquid chromatography with electrochemical detection (HPLC-EC) following enzymatic hydrolysis of DNA or as the base equivalent, 8-oxoguanine, by gas chromatography-mass spectrometry (GC-MS) following acid hydrolysis of DNA. We have noted a discrepancy in the literature whereby the levels of 8-oxo-2'-deoxyguanosine measured by HPLC-EC in UVC-irradiated DNA are significantly higher than when 8-oxoguanine is measured by GC-MS. By making use of the availability of both HPLC-EC and stable-isotope dilution GC-MS methodologies in our laboratory we have confirmed the discrepancy noted in the literature by parallel analysis of the same UVC-irradiated calf thymus DNA samples. Furthermore, analysis of the UVC-induced product by UV-visible spectrophotometry, voltammetry and its detection by a monoclonal antibody which recognises 8-oxo-2'-deoxyguanosine strongly suggests that the product is indeed 8-oxo-2'-deoxyguanosine. Partial explanation for this discrepancy could be an inordinate resistance of UVC-irradiated DNA to formic acid hydrolysis. However, we cannot completely exclude the possibility that there is a formic acid-labile species which co-elutes with 8-oxo-2'-deoxyguanosine in enzymatically digested UVC-irradiated DNA. Whether this phenomenon is unique to UV-irradiation damage or occurs with other systems that cause oxidative damage to DNA awaits further investigation. Irrespective of the exact mechanism, there will be significant implications for the analysis of oxidative DNA damage.

8-Hydroxy-2'-Deoxyguanosine↗