An update on oral cavity cancer: epidemiological trends, prevention strategies and novel approaches in diagnosis and prognosis.

In the UK, the incidence of oral cavity cancer continues to rise, with an increase of around 60% over the past 10 years. Many patients still present with advanced disease, often resulting in locoregional recurrence and poor outcomes, which has not changed significantly for over four decades. Changes in aetiology may also be emerging, given the decline of smoking in developed countries. Therefore, new methods to better target prevention, improve screening and detect recurrence are needed. High-throughput 'omics' technologies appear promising for future individual-level diagnosis and prognosis. However, given this is a relatively rare cancer with significant intra-tumour heterogeneity and variation in patient response, reliable biomarkers have been difficult to elucidate. From a public health perspective, implementing these novel technologies into current services would require substantial practical, financial and ethical considerations. This may be difficult to justify and implement at present, therefore focus remains on early detection using new patient-led follow-up strategies. This paper reviews the latest evidence on epidemiological trends in oral cavity cancer to help identify at risk groups, population-based approaches for prevention, in addition to potential cutting-edge approaches in the diagnosis and prognosis of this disease.

Challenges and novel approaches for investigating molecular mediation.

Understanding mediation is useful for identifying intermediates lying between an exposure and an outcome which, when intervened upon, will block (some or all of) the causal pathway between the exposure and outcome. Mediation approaches used in conventional epidemiology have been adapted to understanding the role of molecular intermediates in situations of high-dimensional omics data with varying degrees of success. In particular, the limitations of observational epidemiological study including confounding, reverse causation and measurement error can afflict conventional mediation approaches and may lead to incorrect conclusions regarding causal effects. Solutions to analysing mediation which overcome these problems include the use of instrumental variable methods such as Mendelian randomization, which may be applied to evaluate causality in increasingly complex networks of omics data.

Survey of malathion resistance and avermectin susceptibility in field populations of Drosophila melanogaster (Diptera: Drosophilidae) and D. simulans.

Populations of Drosophila melanogaster (Meigen) and Drosophila simulans (Sturtevant) from various geographic locations in North America and elsewhere were sampled to assess the distribution of malathion resistance and avermectin tolerance. Comparisons are made to previous reports of a differential response to malathion selection in these species. Results indicate that for malathion, resistance is widespread in D. simulans but varies considerably in D. melanogaster. For avermectins, although the pattern of tolerance is similar in both species, there exists a significant amount of variation in these levels between geographic regions. We consider how these different geographic patterns of resistance distribution may shed insight on the relative roles of population structure and exposure history in affecting the spread of resistance. In addition, we consider further the use of D. melanogaster and D. simulans as a model for examining the effects of insecticide exposure on sympatric populations.

Divergent and conserved features in the spatial expression of the Drosophila pseudoobscura esterase-5B gene and the esterase-6 gene of Drosophila melanogaster.

The regulatory regions of homologous genes encoding esterase 6 (Est-6) of Drosophila melanogaster and esterase 5B (Est-5B) of Drosophila pseudoobscura show very little similarity. We have undertaken a comparative study of the pattern of expression directed by the Est-5B and Est-6 5'-flanking DNA to attempt to reveal conserved elements regulating tissue-specific expression in adults. Esterase regulatory sequences were linked to a lacZ reporter gene and transformed into D. melanogaster embryos. Est-5B, 5' upstream elements, give rise to a beta-galactosidase expression pattern that coincides with the wild-type expression of Est-5B in D. pseudoobscura. The expression patterns of the Est-5B/lacZ construct are different from those of a fusion gene containing the upstream region of Est-6. Common sites of expression for both kinds of constructs are the third segment of antenna, the maxillary palps, and salivary glands. In vitro deletion mutagenesis has shown that the two genes have a different organization of regulatory elements controlling expression in both the third segment of antenna and maxillary palps. The results suggest that the conservation of the expression pattern in genes that evolved from a common ancestor may not be accompanied by preservation of the corresponding cis-regulatory elements.

Malathion resistance levels in sympatric populations of Drosophila simulans (Diptera: Drosophilidae) and D. melanogaster differ by two orders of magnitude.

Malathion resistance levels were determined in populations of the sympatric species Drosophila melanogaster (Meigen) and Drosophila simulans (Sturtevant) from a collection site in Tampa Bay, FL, with a 20+ yr history of intensive malathion exposure. Bioassays of insecticide resistance were done by using a desiccation technique that reduces both the effects of avoidance behavior on the results and the total time of the assay. Resistance levels in isofemale lines of D. simulans are as much as 2 orders of magnitude higher than those in the D. melanogaster lines and are significantly higher than any previously reported resistance levels for organophosphate exposure in Drosophila sp. Comparison with specimens from additional collection sites in the region indicates that the normal ratio of these sympatric species has been altered at our study site. The use of Drosophila sp. as a model system to study resistance management strategies and differences in the evolution of insecticide resistance among sympatric species is discussed.

Platinum drug delivery and radiation for locally advanced prostate cancer.

Combined therapies of cisplatin and radiation have resulted in clinical reports of apparent efficacious control of locoregional cancer and enhanced survival. Mechanisms of interaction between platinum and radiation that may explain these clinical observations all have in common the prediction that higher concentrations of platinum in all tumor cells close in time to irradiation should lead to greater potentiation of radiation-induced killing of those cells. Cisplatin is thus viewed as providing some radiation-equivalent, or a radiation dose-effect factor, for sterilization of tumors. One disease site that has not been well investigated for response to cisplatin plus radiation therapy, but that could benefit from it, is locally advanced prostate cancer. A body of literature now supports the view that local control of stage C (T3, N0, M0) prostate cancer is correlated with disease-free survival. This correlation makes prostate cancer a candidate for potentially achieving improved cure rates following local tumor sterilization by combining cisplatin with radiation therapy. The need and approaches to optimize delivery of cisplatin within tumor tissue is explored. Increasing cisplatin concentration to all the cells of a tumor, i.e., homogeneously delivering systemic high-dose cisplatin, should benefit the efficacious response otherwise expected for cisplatin combined with radiation. Strategies to increase the homogeneity of cisplatin delivery to a tumor are considered to be those that increase perfusion to that tumor. Vasoactive agents used in anticancer protocols are especially considered for their potential value in serving to increase tumor perfusion. These protocol-inclusive agents include certain cytokines and L-arginine antagonists, and should be better managed and accepted in practice compared to other vasoactive agents that need to be developed as specific additives to protocol designs.

Localization of sequences controlling the spatial, temporal, and sex-specific expression of the esterase 6 locus in Drosophila melanogaster adults.

The esterase 6 gene (Est-6) of Drosophila melanogaster is expressed in a variety of tissues that differ between larval and adult stages and among related species. Variability in the level of expression of this locus among different species and strains and its species- and tissue-specific patterns of expression make it a useful system for studying the evolution of gene regulation in Drosophila. We have begun to determine the location of the regulatory regions of Est-6 by constructing deletion mutants of the 5' regions of the gene and transforming them back into flies. Deletion mutants of the putative 5' promoter regions of Est-6 were fused to the bacterial beta-galactosidase gene (lacZ) and assayed for their ability to direct tissue-specific expression in transformed D. melanogaster adults. We have identified four independently acting Est-6 regulatory regions that direct the expression of lacZ in (i) the ejaculatory duct; (ii) the adult salivary glands; (iii) the respiratory system, prefrons, antennae, and maxillary palps; and (iv) the ejaculatory bulb and prefrons. We also found a region near the start of transcription that directed expression of Est-6 in the cardia or proventriculus in some transformed lines.

Effective photodynamic action by rhodamine 123 leading to photosensitized killing of Chinese hamster ovary cells in tissue culture and a proposed mechanism.

The effectiveness of rhodamine 123 (R123) as a photosensitizer of cell killing is relatively low and correlates with its inefficient production of singlet oxygen. The known selective retention of R123 in the mitochondria of epithelially derived carcinoma cells, however, is a selective feature that could lead to a more useful therapeutic ratio if photosensitizing effectiveness could be increased. Chinese hamster ovary (CHO) cells in tissue culture were therefore exposed to R123 shortly before and during illumination under conditions controlled for oxygen concentration and temperature. Effective photosensitization of cell killing, as judged by colony formation, was produced by 95% but not by 19% O2 during illumination of cells at 5 degrees C or 37 degrees C, and this was additionally enhanced at the sublethal temperature of 42 degrees C. Two CHO cell lines were examined; one line, CHO-AA8, was proficient in the repair of DNA damage and the parent to the second line, CHO-EM9, that was deficient in the repair of DNA strand breaks. Cells of both lines incorporated R123 to a similar degree and were similarly photosensitized by the presence of high oxygen concentration. Furthermore, plasma membrane damage as judged by the exclusion of trypan blue was not observed immediately after illumination in the presence of R123, but was seen in the presence of meso-tetra-(4-sulfonatophenyl)-porphine (TPPS4). The extent of damage to the plasma membrane by TPPS4 was greater in the presence of 95% compared to 19% O2 during illumination.(ABSTRACT TRUNCATED AT 250 WORDS)

An evolutionary model for the duplication and divergence of esterase genes in Drosophila.

The esterase 5 (Est-5 = gene, EST 5 = protein) enzyme in Drosophila pseudoobscura is encoded by one of three paralogous genes, Est-5A, Est-5B, and Est-5C, that are tightly clustered on the right arm of the X chromosome. The homologous Est-6 locus in Drosophila melanogaster has only one paralogous neighbor, Est-P. Comparisons of coding and flanking DNA sequences among the three D. pseudoobscura and two D. melanogaster genes suggest that two paralogous genes were present before the divergence of D. pseudoobscura from D. melanogaster and that, later, a second duplication occurred in D. pseudoobscura. Nucleotide sequences of the coding regions of the three D. pseudoobscura genes showed 78-85% similarity in pairwise comparisons, whereas the relatedness between Est-6 and Est-P was only 67%. The higher degree of conservation in D. pseudoobscura likely results from the comparatively recent divergence of Est-5B and Est-5C and from possible gene conversion events between Est-5A and Est-5B. Analyses of silent and replacement site differences in the two exons of the paralogous and orthologous genes in each species indicate that common selective forces are acting on all five loci. Further evidence for common purifying selective constraints comes from the conservation of hydropathy profiles and proposed catalytic residues. However, different levels of amino acid substitution between the paralogous genes in D. melanogaster relative to those in D. pseudoobscura suggest that interspecific differences in selection also exist.

Platinum levels and clinical responses of tumours treated by cisplatin with and without concurrent hyperthermia: a case study.

The heterogeneity of platinum distributed within tissue after clinical administration of cisplatin was evaluated for the first time. Platinum levels were correlated with the observed clinical responses of separate superficial histiocytic sarcomas located in the forearm of a 74-year-old male patient. One of four lesions received four weekly treatments with hyperthermia administered concurrently with 30 mg/m2 cisplatin, while three lesions were treated with cisplatin alone. The lesion receiving hyperthermia concurrently with cisplatin had a solid partial response during a 6-week period following this therapy, whereas two other tumours receiving cisplatin alone progressed. One lesion could not be clinically evaluated. Platinum levels were determined in multiple samplings from three of the four lesions and normal tissue in order to evaluate the validity of taking a single tumour sample of 100 mg or less for the analysis of platinum content. Such a small single sample might provide a value significantly different from the true average because of sampling error. The range in platinum distribution encompassed an average of three-fold difference within eight separate sample groups, with a factor of six being the greatest difference in a single sample group. This degree of heterogeneity is great enough to suggest that conclusions made from the analysis of small and single random tissue samples could be sufficiently in error to misdirect investigative or medical decisions.

An interrelatedness of the potentiation of radiation-induced bacterial cell killing by cisplatin and binuclear rhodium carboxylates.

Cisplatin and binuclear rhodium (Rh2) carboxylates appear to potentiate radiation-induced killing of Salmonella typhimurium cells largely as a consequence of one-electron reduction that leads to an active radiolytic product. This conclusion is supported by results from experiments wherein the hydrated electron and the hydroxyl radical are competed for in the presence of cisplatin and Rh2 carboxylates, and by the similarly shaped radiation survival curves for cisplatin and Rh2 carboxylates wherein potentiation is expressed beyond variable thresholds of radiation dose. Increasing concentrations of phosphate and chloride also inhibit radiation potentiation by both cisplatin and Rh2 carboxylates, further supporting the contention for similar mechanisms. Radiation potentiation by cisplatin is relatively much more sensitive to the inhibition by chloride.

Physical mapping of the Esterase-6 locus of Drosophila melanogaster.

The Esterase-6 gene locus of Drosophila melanogaster although well-characterized, has not been definitely mapped by in situ hybridization. In this paper, a high resolution in situ hybridization protocol using an avidin/biotinylated-horseradish peroxidase/diaminobenzidine system was adopted to refine the physical map position of the Esterase-6 locus. Clarity of signal, detail of banding pattern and absence of background allowed the assignment of a 1.8 kb cDNA encoding Esterase-6 to three bands within subsections 69 A1-A3 on the left arm of polytene chromosome 3. These data refine earlier deletion mapping and low resolution in situ hybridization results, which assigned Esterase-6 to 69A1-A5. The potential use of this high resolution in situ hybridization technique in the analysis of the physical organization of the Esterase-6 gene duplication and surrounding region is discussed.

Cloning of the esterase-5 locus from Drosophila pseudoobscura and comparison with its homologue in D. melanogaster.

A clone of the esterase-5 (Est-5) gene from Drosophila pseudoobscura has been isolated by hybridization to the cloned Est-6 gene of D. melanogaster. Southern analysis and sequencing of the cloned DNA revealed three regions of similarity to Est-6 that have been tentatively identified as genes, Est-5A, Est-5B, and Est-5C. Introduction of each of the three genes separately into D. melanogaster by P-element transformation has demonstrated that Est-5B encodes an enzyme with the same physical properties as EST 5 in D. pseudoobscura. Sequence analysis indicates that Est-5B encodes a 545-amino-acid protein and is composed of two exons separated by a 55-bp intron in the same position as the 51-bp intron in Est-6. Comparison of the Est-5B coding region with that of Est-6 reveals an overall similarity (73% at both the nucleotide and amino acid levels) that is substantially lower than that for other genes sequenced in both of these species. Total nucleotide and nonsynonymous site differences between Est-6 and Est-5B are more abundant in the second exon than in the first, suggesting differential effects of selection or mutation on these two exons. Comparisons of the 5'-flanking DNA of Est-5B and Est-6 reveal four short conserved sequence elements, but the remaining upstream sequences show no significant similarity. Conservation in the 3'-flanking DNA is limited to the presence of two polyadenylation sites that may correlate with the existence of two transcripts from both Est-5B and Est-6. The patterns of nucleotide substitutions and amino acid replacements between Est-5B and Est-6 are consistent with the hypothesis that mutation and genetic drift are responsible for the differences between these two genes.

Molecular analysis of evolutionary changes in the expression of Drosophila esterases.

Drosophila melanogaster transformed with the esterase 5 (Est-5) gene from Drosophila pseudoobscura were used to assess the evolutionary basis for differences in the sex- and tissue-specific expression of the esterase 5 (EST 5) enzyme in D. pseudoobscura relative to its homologue in D. melanogaster, EST 6. EST 5 is expressed in the eyes and hemolymph of transformed D. melanogaster just as it is in D. pseudoobscura, but it is not detectable in the ejaculatory duct, where the homologous enzyme, EST 6, is most abundant. EST 5 also occurs at equal levels in both sexes of the transformants and D. pseudoobscura, whereas EST 6 is more abundant in male than in female D. melanogaster. Northern analysis of transformed and untransformed flies indicates that the expression patterns of EST 5 and EST 6 are controlled at the level of transcription and suggests that regulatory differences between Est-6 and Est-5 have evolved mainly through cis-acting regulatory changes in the two loci rather than through alterations in trans-acting factors. Equal expression of EST 5 in male and female transformants also indicates that the X-chromosome-linked Est-5 gene of D. pseudoobscura, when isolated as a 4.5-kilobase restriction fragment, is not dosage compensated after integration into an autosome of D. melanogaster.

Molecular analysis of duplicated esterase genes in Drosophila melanogaster.

Genomic clones containing sequences homologous to an esterase 6 (Est-6) cDNA clone were isolated from a library of Drosophila melanogaster DNA. Comparison of the genomic and cDNA sequences revealed that the Est-6 gene comprises two exons, one of 1,387 bp and one of 248 bp, separated by a short intron of 51 bp. Further sequencing revealed the presence of a tandem duplication of the Est-6 gene (denoted Est-P) which also has an exon of 1,387 bp and an exon of 248 bp, separated by a short intron of 56 bp. The two genes show similarities of 64% and 60% at the DNA and protein levels, respectively. The coding regions of the genes are 197 bases apart, and presumptive 5' regulatory sequences of Est-P overlap at least the 3' noncoding region of Est-6. Transcripts homologous to Est-P were detected in late larvae and adults of each sex, whereas Est-6 transcripts are present in all life stages but are predominant in adult males. This suggests different physiological functions for the products of the two genes. Southern and Northern blot hybridization analyses of the 20-kb region surrounding the Est-6/Est-P duplication failed to detect any other duplicated esterase genes, although this region is actively transcribed.

Potentiation of radiation-induced bacterial cell killing by binuclear rhodium(II) carboxylates.

A series of binuclear rhodium(II) tetracarboxylate complexes was examined for potentiation of radiation-induced killing of Salmonella typhimurium cells. Carboxylate bridging ligands were varied as formate, acetate, trifluoroacetate, and propionate. All complexes caused hypoxic non-dose-modifying radiation potentiation in that variable thresholds were obtained with the radiation dose response. In phosphate-buffered saline (PBS), decreasing threshold doses, i.e., increasing potentiating efficiencies, were seen in the order of acetate = trifluoroacetate less than propionate less than formate. Beyond the threshold dose, the degree of potentiation for all complexes in PBS approximated 12 times the degree of radiation sensitivity seen for the N2 baseline of the radiation dose-response curve. No radiation potentiation by Rh2 carboxylates was seen for fully oxic suspensions. Irradiation of cells in the absence of phosphate increased the efficiency as well as the degree of radiation potentiation. It is hypothesized that bacterial radiation potentiation is initiated by one-electron reduction of the Rh2 carboxylates, most likely involving the hydrated electron, followed then by formation of an active product. These events likely occur outside the bacterial cell.

Potentiation of radiation-induced bacterial cell killing by binuclear rhodium(II) complexes and their amines.

Rhodium(II) binuclear complexes were surveyed for potentiation of radiation-induced cell killing of hypoxic and fully oxic Salmonella typhimurium cells. The Rh2 tetracarbonate ion substantially potentiated hypoxic cell radiation sensitivity. Phosphate interfered with this potentiation. In the latter two respects, radiation potentiation by Rh2 tetracarbonate is similar to that found for Rh2 tetracarboxylates. Amines such as ammonia, methylamine, ethylamine, n-propylamine, and n-butylamine were examined with both Rh2 tetracarbonate and tetraacetate complexes. With Rh2 tetraacetate in phosphate-buffered saline, these amines variably increased radiation potentiation to a maximum of nearly that seen by Rh2 tetraacetate alone in the absence of phosphate. With Rh2 tetracarbonate, particular amines were found to either enhance or restrict radiation potentiation. Results as a whole support the hypothesis that a radiolytic Rh species initiated in a one-electron reduction process external to the cell is responsible for the potentiation by Rh2 complexes in bacteria. Phosphate interference of potentiation by Rh2 tetracetate appears to be limited competitively by amines, suggesting that axial associations of phosphate with the Rh2 center may be involved in the inhibition of radiation potentiation. Of interest in this regard is the finding that 5'-adenosinemonophosphate eliminates the potentiation seen with Rh2 tetraacetate.

Biologic and clinical developments of cisplatin combined with radiation: concepts, utility, projections for new trials, and the emergence of carboplatin.

Controlled experiments have shown that more than one mechanism leads to the potentiation of radiation-induced cell killing by cisplatin, and that this potentiation is not uniformly expressed among different cell types. A firm investigative base for the design of clinical trials using cisplatin and radiation has not been established. Coincident with this deficiency of experimental guidance, the independent clinical investigator has developed an array of therapeutic strategies applying different doses and sequences of cisplatin and radiation to a variety of tumor types. Results of clinical studies integrating cisplatin and radiation that can be judged for perceived survival benefit are evaluated in comparison with existing radiobiologic information. Both the clinical and radiobiologic results lead to similar conclusions at this time. Cells that are relatively sensitive to the cytotoxic action of cisplatin alone would best be considered for combined treatment with radiation. Large and infrequent, rather than small and frequent, individual administrations of cisplatin are better used with radiation for enhanced therapeutic effectiveness. Administration of cisplatin close in time to radiation is best for therapeutic response, although perceived efficacy follows from rather flexible integrations of these two modalities. It is not possible to know if clinical efficacy results from radiation potentiation as opposed to some degree of additivity of the two modalities. It is nonetheless useful to anticipate strategies that might lead to radiation potentiation by cisplatin in therapeutic designs. Two general mechanisms by which cisplatin potentiates radiation-induced cell killing are identified. One mechanism of potentiation is free radical-mediated, at least in part leads to an active radiolytic species following one-electron reduction of cisplatin, and is more readily expressed with bacterial cells than with mammalian cells in tissue culture. A second mechanism of potentiation is biochemical in nature, involves an effect of cisplatin on cellular components in ways that inhibit the recovery of radiation-induced damage, and likely applies more to the potentiation of oxic mammalian cells than bacterial cells. The latter mechanism is not universally supported in the literature. However, a unifying hypothesis, and one in need of confirmation at this time, is that the biochemical mechanism of radiation potentiation by cisplatin operates in oxic mammalian cells that are inherently sensitive to the cytotoxic action of cisplatin. This hypothesis ostensibly applies to tumor cells that are responsive to chemotherapy with cisplatin.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular population genetics of structural variants of esterase 6 in Drosophila melanogaster.

Several lines of evidence indicate that natural selection operates between the major EST6-F and EST6-S allozymes of Drosophila melanogaster. In particular, consistent latitudinal clines and seasonal variation in their relative frequencies strongly suggest that they are not selectively equivalent in field populations. Several laboratory studies have found frequency-dependent fitness differences among the Est6-F and Est6-S genotypes. Moreover, the purified EST6-F and EST6-S allozymes differ in biochemical properties and the physiology of the enzyme, as a major component of the seminal fluid, suggests that these differences could affect reproductive aspects of fitness. However, molecular analyses reveal high levels of variation in the EST6 protein both within and between the EST6-F and EST6-S allozymes. Limited thermostability and more sensitive electrophoretic analyses reveal at least 17 variants of the two allozymes and sequence comparisons among 13 isolates of the Est6 gene reveal 16 nucleotide polymorphisms that would lead to amino acid differences. Two closely linked amino acid differences are strongly associated with the major difference between EST6-F and EST6-S; either or both of these are likely to cause the observed biochemical differences between EST6-F and EST6-S and may be the primary targets for the selection between these allozymes. The functional and adaptive significance of the other amino acid polymorphisms is unclear, although the data suggest that the EST6-8 haplotype within EST6-S has both arisen and proliferated relatively recently.