Detection of minimal residual cancer to investigate why oral tumors recur despite seemingly adequate treatment.

Improvements in surgery and radiotherapy techniques have led to only a modest increase in the 5-year survival rate for patients with head and neck cancer. This is because the pattern of clinical disease is changing, such that locoregional recurrence now accounts for fewer treatment failures, but more patients develop a second primary cancer or distant metastatic disease. In this study, we have used the p53 phage plaque assay, immunocytochemistry, and mutational analysis to assess the contribution of minimal residual cancer and genetic aberrations in clinically normal upper aerodigestive tract mucosa to treatment failure. Eighteen consecutive patients with oral tumors, with conventional clear margins, have been followed for a minimum of 36 months. Molecular assessment identified tumor-positive surgical margins for 6 of 11 assessable patients and additional tumor-positive lymph nodes for three cases. Disseminated malignant cells were detected in the hematopoietic cell compartment for six cases, and one patient had molecular evidence of field cancerization. Locoregional recurrence developed in five patients with tumors harboring a p53 gene mutation; four of these were associated with tumor-positive surgical margins, and one was associated with molecular evidence of field cancerization. Radiotherapy to the primary site did not prevent development of local recurrence when the residual tumor harbored a p53 gene mutation. Three of six cases with a tumor-positive bone marrow aspirate developed distant metastases. These findings reveal that molecular and immunocytochemical detection of minimal residual cancer and field cancerization can help identify patients who may develop locoregional or distant recurrence and justify further studies to evaluate the contribution of these remaining malignant cells to treatment failure.

Xanthine oxidoreductase in human mammary epithelial cells: activation in response to inflammatory cytokines.

Xanthine oxidoreductase (XOR) in human mammary epithelial cells was shown to have low true specific activity, similar to that in breast milk. Enzymic activity was increased in response to inflammatory cytokines; increases of 2-2.5-fold being seen with TNF-alpha and IL-1beta and of approximately 8-fold with IFN-gamma. No significant increase was seen with IL-6. A combination of IFN-gamma and TNF-alpha, or of these two cytokines plus IL-1beta, led to responses representing the sum of those obtained by using the individual cytokines. The 8-fold increase in enzymic activity, stimulated by IFN-gamma, corresponded to only a 2-3-fold increase in specific mRNA, suggesting the possibility of post-translational activation; a possibility strongly supported by the corresponding 2-3-fold rise in XOR protein, as determined by ELISA. In no case was cytokine-induced activation accompanied by changes in the oxidase-dehydrogenase ratio of XOR. These data strongly support a role for XOR in the inflammatory response of the human mammary epithelial cell, and provide further evidence of post-translational activation of a low activity form of human XOR, similar to that previously observed in vivo for the breast milk enzyme.

Specific binding of DnaA protein to a DnaA box in the guaB gene of Escherichia coli K12.

Expression of the guaBA operon of Escherichia coli is regulated by the DNA replication-initiating protein, DnaA. Two DnaA boxes, which are potential binding sites for DnaA, are present in the gua operon. One box (with 8/9 match to the DnaA box consensus sequence) is at the gua promoter; the other box, which has a consensus sequence, is on the non-transcribed strand within the guaB coding region approximately 200 bp downstream of the initiation codon. The binding in vitro of purified DnaA protein to these boxes was investigated by filter retention and gel retardation analysis, and by deoxyribonuclease I footprinting, using restriction fragments of gua operon DNA. DnaA protein was shown to bind specifically only to the fragment carrying the consensus sequence DnaA box, and to protect this box from deoxyribonuclease I. Transcription termination resulting from the binding of DnaA to this box within the guaB gene explains repression by DnaA of the gua operon in vivo.

Superoxide dismutase of Cryptococcus neoformans: purification and characterization.

We have purified to homogeneity a putative superoxide dismutase of 19.5 kDa from the pathogenic yeast Cryptococcus neoformans by homogenization, isoelectric focusing and gel filtration. The N-terminal amino acid sequence of this protein indicates a significant sequence homology with known manganese-containing superoxide dismutases (Mn-SODs) from various organisms. In addition, the presence of superoxide dismutase activity was confirmed using specific substrate gels which detect this enzyme when nitro-tetrazolium blue reduction is prevented by the photochemical source of superoxide, in the presence of riboflavin when exposed to light. Superoxide dismutase activity was also assayed using cytochrome c. The molecular weight of the native enzyme (on non-denaturing gels) is 80 kDa. The optimum pH for the enzyme is 7.5 and its pi = 6.6. The enzyme was inhibited by sodium dodecyl sulphate, sodium azide, o-phenanthroline, and EDTA, in descending order.

Regulation of the gua operon of Escherichia coli by the DnaA protein.

The guaBA operon determines production of the two enzymes required to convert hypoxanthine to guanine at the nucleotide level during guanine nucleotide biosynthesis. Two DnaA boxes, binding sites for the DNA replication-initiating DnaA protein, are present in the gua operon, one at the gua promoter (guaP) and the other within the guaB coding sequence. Regulation of the guaBA operon by DnaA protein was studied using strains carrying chromosomal gua-lacZ fusions. In these strains beta-galactosidase acts as a reporter enzyme for transcription initiated at guaP. When the intracellular levels of DnaA were increased (by induction of a multicopy plasmid carrying the dnaA gene fused to the tac promoter) transcription from the gua promoter was repressed. Reducing the intracellular level of DnaA, either by sequestration with an oriC plasmid or by placing a temperature-sensitive dnaA mutant at the restrictive temperature, resulted in increased transcription from guaP. Thus the transcriptional activity of the gua operon is coupled, through the DnaA protein, to the DNA replication cycle. Repression of guaP by DnaA was dependent on the presence of both boxes in the gua-lacZ fusion; constructs containing only the box at guaP were unaffected by DnaA.