Mitochondrial DNA mutations in head and neck cancer are infrequent and lack prognostic utility.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations occur in head and neck squamous cell carcinoma (HNSCC) and are most frequently detected in the displacement-loop (D-loop) region. The D-loop is considered to be important because it controls mitochondrial gene expression and mtDNA replication. There is currently no evidence that mtDNA mutations can be used as prognostic or predictive biomarkers in HNSCC. METHODS: We used denaturing high performance liquid chromatography to screen the entire mitochondrial genome of six oral squamous cell carcinoma-derived cell lines and then focused on detecting D-loop abnormalities in 34 HNSCC tissue samples. RESULTS: Mitochondrial DNA mutations are not ubiquitous in HNSCC because only half of the cell lines had detectable mtDNA abnormalities following screening of the entire mitochondrial genome and only 18% (6 of 34) of tissue samples had D-loop mutations. There was no correlation between D-loop mutations and determinates of clinical outcome; specifically, tumour stage and the expression of hypoxia-inducible genes included in a highly prognostic hypoxia metagene. CONCLUSIONS: Taken together, these data suggest that mtDNA D-loop mutations are stochastic events that may not significantly influence the biology of HNSCC and supports the hypothesis that mtDNA mutations in cancer represent bystander genotoxic damage as a consequence of tumour development and progression.

The predictive value of p53 and p33(ING1b) in patients with Dukes'C colorectal cancer.

OBJECTIVE: Identification of biological markers that may predict response to chemotherapy would allow the individualization of treatment by enabling selection of patients most likely to benefit from chemotherapy. The aims of this study were to determine whether p53 mutation status and p53 and p33(ING1b) protein expression can predict which patients with Dukes' C colorectal cancer following curative surgical resection respond to adjuvant chemotherapy with 5-fluorouracil (5-FU). METHOD: Patients with Dukes'C colorectal cancer (n = 41) were studied. DNA was extracted and analysed for p53 mutation using PCR-based direct DNA sequencing. Tumours were analysed for p53 protein expression by immunohistochemistry using DO-7 monoclonal antibody and for p33(ING1b) protein expression using GN1 monoclonal antibody. RESULTS: There was a significant association between p53 mutation status analysed by gene sequencing and overall and metastasis-free survival (P = 0.03 and 0.004, respectively, log-rank test). By contrast, no significant correlation was found between p53 and p33(ING1b) protein expression and overall or metastasis-free survival. CONCLUSION: In patients with Dukes'C colorectal cancer who underwent curative surgical resection of the primary tumour, followed by 5-FU-based adjuvant chemotherapy, p53 mutation status as assessed by gene sequencing is a significant predictor of overall and metastasis-free survival.

Alterations of TP53 in microdissected transitional cell carcinoma of the human urinary bladder: high frequency of TP53 accumulation in the absence of detected mutations is associated with poor prognosis.

We have used microdissection of paraffin-embedded histological sections and polymerase chain reaction (PCR)-based direct DNA sequencing for 54 transitional cell carcinoma (TCC) of the bladder, to examine critically the association between TP53 nuclear accumulation determined by immunohistochemistry and the presence of TP53 mutations, and to examine their relationship to tumour stage and grade, as well as patient survival. There was a significant association between the presence of TP53-positive nuclei (> 10%) and a higher histological stage and grade (P = 0.0115, P = 0.0151 respectively; Fisher's exact). A significant association between TP53 gene mutations and TP53 nuclear reactivity in more than 10% of tumour cell nuclei was also observed (P = 0.0003; Fisher's exact). Mutations were detected in 18/54 (33%) cases together with the wild-type sequence when analysed from bulk frozen samples, with significant clustering of mutations in exons 7 and 8. The microdissection method distinguished more clearly between heterozygous and/or homozygous alterations of the TP53 tumour-suppressor gene, and clearly showed frequent accumulation of TP53 in the absence of mutations. When microdissecting immunonegative regions from the same paraffin sections, three out of ten samples showed the identical mutations detected in the immunopositive regions. There was a significant association between TP53 immunoreactivity in more than 50% of tumour cell nuclei and decreased survival among all patients (P = 0.0325; log-rank test). The patients with TP53 mutations showed a trend for a shorter survival period; however, the association was not statistically significant at the 95% confidence level (P = 0.132; log-rank test). In conclusion, our observations show that accumulation of TP53 occurs frequently in the absence of mutations, and that such accumulation is nevertheless associated with poor survival when it occurs in a high proportion (> 50%) of tumour cell nuclei.

Analysis of the p53 tumor-suppressor gene in hepatocellular carcinomas from Britain.

Human hepatocellular carcinomas from patients in Britain, an area of low prevalence of hepatocellular carcinoma and low dietary exposure to aflatoxin B1, were analyzed for mutations in the p53 tumor-suppressor gene. Abnormalities in the p53 gene were detected in 2 of 19 hepatocellular carcinomas by polymerase chain reaction--single-stranded conformation polymorphism. Direct sequencing of the evolutionarily conserved regions of p53 (exons 5, 6, 7 and 8), where mutations have been commonly found in a variety of tumors, confirmed that only two hepatocellular carcinomas had mutations in p53, one a 6-bp deletion of codons 158 and 159 (exon 5) and the other a G to A transition at codon 286 (exon 8). No mutations were found in any hepatocellular carcinoma in exons 6 and 7; in particular all tumors had wild-type sequence at codon 249, which has been reported to be a mutational hot spot in the p53 gene in hepatocellular carcinomas from high incidence areas such as China and southern Africa. Abnormalities in p53 expression were examined by immunohistochemistry and found in 1 of the 19 hepatocellular carcinomas. These findings show that p53 mutations are infrequently involved in the malignant transformation of hepatocytes in an area of low hepatocellular carcinoma prevalence. They support the suggestion of a possible link between dietary exposure to aflatoxin and selective G to T mutations at codon 249 of the p53 gene. Our observations also indicate that hepatitis B virus infection alone, present in six of the hepatocellular carcinomas examined, does not account for the specificity for codon 249 mutations reported from endemic areas.

Infrequent point mutations in codons 12 and 61 of ras oncogenes in human hepatocellular carcinomas.

DNA from human hepatocellular carcinomas (HCC) were analysed for the presence of mutations in codons 12 and 61 of the K-ras, H-ras and N-ras genes. The relevant ras sequences were amplified in vitro using the polymerase chain reaction and point mutations detected by selective hybridisation using mutation-specific synthetic oligonucleotides. In one of the 19 HCCs a mutation in codon 61 of the K-ras gene was detected, whilst in 3/19 HCCs a mutation was found in codon 61 of the N-ras gene. The mutations were all heterozygous A-T transversions and were found in HCCs arising in patients with underlying cirrhosis. In two of these patients where the corresponding normal tissue was available only the wild-type ras gene was detected, indicating that oncogenic activation of the ras gene was a consequence of somatic mutation. In another patient the same mutation in codon 61 of the N-ras gene was found in cirrhotic liver tissue and in all four patients the same mutation was also detected in formalin-fixed, paraffin-embedded liver biopsy HCC tissue obtained at diagnosis. These results indicate that mutational activation of the ras genes at codon 61 is an infrequent but possibly early event in the development of HCC in Britain.

The chick embryo fibroblast cytosolic DNA complex--a possible cell-cell messenger.

1. The uptake of [3H]thymidine and [3H]uridine labelled DNA-RNA cytosol complex has been studied in chick embryo fibroblast cells. 2. The complex appears to be cleaved into DNA and RNA containing fragments in the recipient cell nucleus: both then enter the cell cytosol fraction, but the RNA fragment in particular is rapidly degraded. 3. Although [3H]thymidine labelled material present in the nucleus co-extracts with bulk nuclear DNA, caesium gradienting shows little or no evidence that integration of host and imported DNA has occurred. 4. It is suggested that the cytosolic/extruded DNA complex may be a "messenger" DNA, capable of the transfer of regulatory information between cells on a transient basis.

The search for the DNA of the chick embryo fibroblast cytosolic complex.

1. Conventional DNA extraction procedures have failed to release free DNA from the chick embryo fibroblast cytosolic DNA-RNA complexes. 2. Free DNA has been released only from the smallest cell cytosol DNA fraction, which is not in the native state associated with RNA: it is very small (of the order of 100 bases) and single stranded. 3. However, phenol extraction does separate complex DNA-associated material from the RNA which has invariably been found to accompany it in all but the smallest fraction (see 2 above). 4. The principal factor preventing DNA release appears to be a massive aggregation of partially purified DNA-associated material.

The assembly of the DNA complex present in chick embryo cell cytosol.

Chromatography of chick embryo fibroblast cytosol labelled with [3H]thymidine or [3H]uridine precursors has shown the presence of early labelled DNA and RNA eluting at a position corresponding to a relative molecular mass of approximately 1.5-10(5). The early DNA-RNA (heteroduplex?) then moves progressively to a higher molecular weight peak, relative molecular mass approximately 10(6). The process appears similar in cytosol from cultured cells and from whole amniotically labelled chick embryos: consequently the cytosolic DNA complex is not an artefact of cell culturing. The relative contribution of artefactual and specific cytosol-associated DNA material is discussed: it is concluded that while both are present in cytosol as prepared, it is possible to discriminate between specific and artefactual DNA material.

Further studies on the size and composition of the chick embryo fibroblast cytosolic DNA complex.

The chick embryo fibroblast cytosolic DNA complex shows anomalous elution behaviour on agarose gel column chromatography. The indicated molecular size varies between 5 X 10(5) dalton (higher exclusion limit gels) and 1.4 X 10(6) dalton (lower exclusion limit gels). Chromatography on lower exclusion limit gels shows the [3H]thymidine labelled (DNA) complex as a sharp peak, coincident with a peak of [3H]uridine and [3H]lysine labelling and similar pulse labelling patterns for the three precursors but with DNA labelling lagging behind RNA and protein. Both cultured and uncultured cell cytosols show an A260 peak coincident with the [3H]precursor labelling peaks.