Is tooth wear in the primary dentition predictive of tooth wear in the permanent dentition? Report from a longitudinal study.

OBJECTIVES: To determine the prevalence of tooth wear in the permanent dentition of a sample of 12-year-old school children and establish whether an association exists between tooth wear recorded now and tooth wear recorded in their primary dentition at age five. DESIGN: A prospective cohort study. METHODS: At follow-up to a previous study complete data were available for 123 children; fieldwork was conducted in the child's primary school. Measurement of tooth wear used a scoring system modified from the Smith and Knight Tooth Wear Index (TWI). Tooth wear which had progressed to dentine was assessed on the occlusal surfaces of the four first permanent molars, the labial, lingual/palatal and incisal surfaces of the six upper and six lower anterior teeth; a total of 40 scoreable surfaces. Demographic data were collected from the parents, and a questionnaire on oral hygiene habits, diet and behaviours was completed by each child. RESULTS: In total 38% (n = 47) of subjects had tooth wear, if incisor teeth only were included, 33% (n = 40) had tooth wear and similarly if the occlusal surfaces of molar teeth only were included 10% (n = 12) had signs of tooth wear. Gender was significantly associated with tooth wear: males had more tooth wear. The presence of tooth wear with dentine exposed in the primary dentition was significantly associated with tooth wear on the occlusal surfaces of the first permanent molars. CONCLUSION: Males had more tooth wear than females. An association existed between tooth wear recorded at age 5 and molar tooth wear recorded at age 12. Tooth wear is a lifelong cumulative process and should be recorded in both the primary and permanent dentitions.

Primary tooth fluorosis in 5-year-old schoolchildren in Ireland.

AIM: This was to determine the prevalence of primary tooth fluorosis in the dentitions of 5-year-old schoolchildren. A subsidiary aim was to investigate whether an association existed between the presence of primary tooth fluorosis, fluoridation status, infant feeding practices or the oral hygiene practices of the child. STUDY DESIGN: A cross-sectional and stratified by fluoridation status study. METHODS: Fluorosis was recorded using a modification of the Tooth Surface Index of Fluorosis (TSIF). Demographic data, information on infant feeding practices and oral hygiene practices were collected via a parental questionnaire. STATISTICS: Stepwise logistic regression analysis. RESULTS: Fluorosis prevalence in the fluoridated group (n=208) was 32%; 29.3% (n=61) had a modified TSIF score of 1; 2.4% (n=5) had a modified TSIF score of 2; and 1% (n=1) had a modified TSIF score of 5. In the non-fluoridated group (n=86) one child had a modified TSIF score of 1. Primary tooth prevalence of fluorosis in the entire sample (n=294) was 23%. Factors that were associated with primary tooth fluorosis were: fluoridation status (p= 0.0003, 95% CI = 5-281) and the age at which toothbrushing with toothpaste commenced (p = 0.016, 95% C.I. 1.1 - 3.8). No association with infant feeding practices was identified. CONCLUSION: The overall prevalence of primary tooth fluorosis was 23%. Lifetime residence in a fluoridated area and commencement of toothbrushing with toothpaste between 12 and 18 months of age were associated with primary tooth fluorosis. No association with infant feeding practices was identified.

Dental erosion in 5-year-old Irish school children and associated factors: a pilot study.

OBJECTIVE: To determine the prevalence of dental erosion in a stratified sample of 5-year-old children and to investigate whether demographic and dietary factors were associated. DESIGN: Cross sectional study in Cork City and County. METHODS: A sample of 202 5-year-old children stratified on fluoridation status was selected. Measurement of erosion used a scoring system and criteria based on those used in the UK. Wear on the palatal and labial surfaces of primary maxillary teeth considered to be predominantly erosive was assessed. Demographic and dietary details were collected via a parental questionnaire. Statistical analysis was stepwise logistic regression. RESULTS: In lifetime residents of fluoridated areas (n = 114) 47% had evidence of erosion; in 21% erosion had progressed to the dentine or pulp. The corresponding figures in non-fluoridated areas (n = 76) were 43% and 21% respectively. The variables significantly associated with erosion to dentine or pulp were low socio-economic status, measured by low family income and the frequency of fruit squash and carbonated drink consumption. CONCLUSION: The prevalence of dental erosion overall was 47%, in 21% erosion affected the dentine or pulp. Levels in fluoridated and non-fluoridated areas were similar. Low socio-economic status and frequency of fruit squash and carbonated drink consumption were associated with erosion extending to dentine or pulp.

Cell density mediated pericellular hypoxia leads to induction of HIF-1alpha via nitric oxide and Ras/MAP kinase mediated signaling pathways.

Environmental signals in the cellular milieu such as hypoxia, growth factors, extracellular matrix (ECM), or cell-surface molecules on adjacent cells can activate signaling pathways that communicate the state of the environment to the nucleus. Several groups have evaluated gene expression or signaling pathways in response to increasing cell density as an in vitro surrogate for in vivo cell-cell interactions. These studies have also perhaps assumed that cells grown at various densities in standard in vitro incubator conditions do not have different pericellular oxygen levels. However, pericellular hypoxia can be induced by increasing cell density, which can exert profound influences on the target cell lines and may explain a number of findings previously attributed to normoxic cell-cell interactions. Thus, we first sought to test the hypothesis that cell-cell interactions as evaluated by the surrogate approach of increasing in vitro cell density in routine normoxic culture conditions results in pericellular hypoxia in prostate cancer cells. Second, we sought to evaluate whether such interactions affect transcription mediated by the hypoxia response element (HRE). Thirdly, we sought to elucidate the signal transduction pathways mediating the induction of HRE in response to cell density induced pericellular hypoxia in routine normoxic culture conditions. Our results indicate that paracrine cell interactions can induce nuclear localization of HIF-1a protein and this translocation is associated with strong stimulation of the HRE-reporter activity. We also make the novel observation that cell density-induced activity of the HRE is dependent on nitric oxide production, which acts as a diffusible paracrine factor secreted by densely cultured cells. These results suggest that paracrine cell interactions associated with pericellular hypoxia lead to the physiological induction of HRE activity via the cooperative action of Ras, MEK1, HIF-1a via pericellular diffusion of nitric oxide. In addition, these results highlight the importance of examining pericellular hypoxia as a possible stimulus in experiments involving in vitro cell density manipulation even in routine normoxic culture conditions.

The role of vascular endothelial growth factor in the tissue specific in vivo growth of prostate cancer cells.

Despite the fact that cancer cells can be found in many vascular beds, continued growth of the metastatic tumor focus exhibits a significant degree of 'organ tropism', with only certain organs exhibiting the ravages of metastatic disease. Since a limiting factor to the growth of metastases beyond 2 mm in diameter, may be a lack of angiogenesis, we sought to determine whether tumor overexpression of vascular endothelial growth factor (VEGF), a potent angiogenic factor related to prostate cancer metastasis, is causally related to organ specific tumor growth in a prostate cancer xenograft model. LnCaP-C4-2 is a subline of the human prostate cancer cell line LnCaP which unlike its parent, has a predilection for growth in bone, a common site for human prostate cancer metastasis. LnCaP-C4-2, is tumorigenic when injected intrafemorally in mice but requires co-injection of stromal components (Matrigel) to be tumorigenic in the subcutaneous site. Because of this site-specific tumorigenicity profile and relatively low VEGF mRNA and protein expression, this line was transfected with a full length cDNA encoding the 165 isoform of VEGF. Cells either overexpressing or not expressing the transfected gene were selected for study in vivo and in vitro. Overexpression of VEGF did not seem to affect in vitro cell growth. Such overexpression did affect tumorigenicity and in vivo tumor growth rates when cells were inoculated in the subcutaneus site. Interestingly, the dependency of subcutaneous tumorigenicity on Matrigel co-inoculation was still observed in cells overexpressing VEGF. In contrast to the impact that VEGF overexpression has on subcutaneous tumorigenicity, no such effect was observed when cells were inoculated in orthotopic/prostate (primary) or intrafemoral (metastatic) sites. In view of the importance of tumor-stromal interactions in growth of xenografts, we sought to determine if the host strain is important to the observed tumorigenicity effects of VEGF overexpression. No differences in subcutaneous tumorigenicity as a function of either Matrigel use or VEGF expression levels were observed when SCID/bg and RAG/pfp mouse strains were compared. In conclusion, our data indicate that the biological impact of prostate tumor VEGF overexpression is organ/site specific, leading to the speculation that it may play a part in the observed organ tropism of metastatic spread. In addition, these results highlight the importance of the tumor microenvironment in determining the biological impact of transfected and overexpressed genes in the study of tumor biology.

Transmembrane motility assay of transiently transfected cells by fluorescent cell counting and luciferase measurement.

Current in vitro assays used in assessing tumor motility could be improved by the development of a simple technique that would facilitate studies of the impact of specific genes on pharmacologically altered chemotaxis. We developed a technique that improves on the classic transwell assay by using fluorescence and luminescence to assess chemotaxis. In this transient transfection system, co-transfection of a reporter construct and a gene with an unknown impact on motility are coupled with biochemical assays to quantitate the number of cells that have received a transferred gene, which subsequently crosses the membrane. This assay was found to be less variable than the conventional transwell chamber and is easily adaptable to studies of cell motility or cell invasion. We also demonstrate that this assay can detect the effect of both genetic and pharmacological inhibition of motility alone and in combination. It therefore has the potential to reveal additive or synergistic effects.

Focal adhesion kinase, Rap1, and transcriptional induction of vascular endothelial growth factor.

BACKGROUND: Signals from a cell's environment are sensed by receptors, which activate pathways that, in turn, transmit the signals to the nucleus, informing decisions on growth, angiogenesis, and other cell functions. Transcription of vascular endothelial growth factor (VEGF), a potent angiogenic factor, can be induced by cell-cell contact. In the current work, we sought to determine if this induction is dependent on transformation of cells to a malignant phenotype and subsequently to determine which signaling molecules mediate activation of VEGF transcription. METHODS: Normal and transformed prostate epithelial cell lines were examined at various cell densities to simulate the effect of increased cell contact on expression of VEGF messenger RNA. Transformed cells were also cotransfected with a VEGF promoter-reporter construct and with constructs that express dominant negative or activated versions of signal transduction proteins hypothesized to be involved in the cell-cell contact process, and reporter activity was assessed at various cell densities. All P values are two-sided. RESULTS: Direct cell-cell contact, but not extracellular matrix components, resulted in transcriptional activation of a VEGF promoter-reporter construct in malignant (P<.0001) but not in nonmalignant (P =.37) prostate cells. This process was mediated via a mitogen-activated protein kinase (MAPK); it required the activity of focal adhesion kinase (FAK), Rap1, and Raf and was Ras independent. In addition, transcriptional activation of a Ras-sensitive Elk-1 chimeric reporter by cell-cell contact suggests that Rap1 is a key factor in regulating the specificity of convergent MAPK-signaling pathways arising from different upstream extracellular stimuli. CONCLUSIONS: Cell contact induction of VEGF transcription via FAK and Rap1 provides a novel Ras-independent, but transformation-dependent, mechanism for stimulus-specific regulation of tumor VEGF expression via MAPK.

Molecular cloning and characterization of a lysophosphatidic acid receptor, Edg-7, expressed in prostate.

Two G protein-coupled receptors (Edg-2) and (Edg-4) for the lysolipid phosphoric acid mediator lysophosphatidic acid have been described by molecular cloning. However, the calcium-mobilizing receptor Edg-4 is not expressed in some cell lines that exhibit robust calcium responses to this ligand, thus predicting the existence of additional receptor subtypes. We report here on the characterization of a third human lysophosphatidic acid receptor subtype, Edg-7, which mediates lysophosphatidic acid-evoked calcium mobilization. In a rat hepatoma Rh7777 cell line that lacks endogenous responses to lysophosphatidic acid, this lipid mediator, but not others, evokes calcium transients when the cells have been transfected with Edg-7 or Edg-4 DNAs. Furthermore, frog oocytes exhibit a calcium-mediated chloride conductance in response to mammalian-selective lysophosphatidic acid mimetics after injection of Edg-7 mRNA. Edg-7-expressing Rh7777 cells do not show inhibition of forskolin-driven rises in cAMP in response to lysophosphatidic acid. However, membranes from HEK293T cells cotransfected with Edg-7 and G(i2)alpha protein DNAs show lysophosphatidic acid dose-dependent increases in [gamma-(35)S]GTP binding with an EC(50) value of 195 nM. When we used this assay to compare various synthetic LPA analogs at Edg-2, Edg-4, and Edg-7 receptors, we found that ethanolamine-based compounds, which are full LPA mimetics at Edg-2 and Edg-4, exhibit little activity at the Edg-7 receptor. Edg-7 RNA was detected in extracts of several rat and human tissues including prostate. Together, our data indicate that Edg-7 is a third lysophosphatidic acid receptor that couples predominantly to G(q/11)alpha proteins.

Genetic and phenotypic changes associated with the acquisition of tumorigenicity in human bladder cancer.

There has been a general lack of human paired cell lines that both reproduce the in vivo spectrum of tumor progression of bladder cancer and have some of the genetic changes associated with progression in human tumor tissue. T24, a cell line established from an invasive human transitional cell carcinoma (TCC) of the bladder, has been used extensively in bladder cancer research. However, a significant limitation of this cell line is its lack of tumorigenicity when injected into immunocompromised mice. This characteristic was used to our advantage as we sought to characterize T24T, a highly tumorigenic variant that could then be used to elucidate the genes responsible for human bladder tumor progression. In culture, T24T has a faster doubling time, reaches a higher cell density in monolayer culture, and is more motile than T24 at higher cell densities. T24T is able to form colonies in soft agar, whereas T24 is not, and expresses HRAS, a gene associated with increased aggressiveness in human TCC, at higher levels than T24. Most importantly, T24T forms solid tumors when injected subcutaneously in SCID mice both with and without Matrigel (Sigma, St. Louis, MO), whereas T24 does not. Cytogenetically, the 2 cell lines contain at least 5 shared structural anomalies, as determined by detailed karyotyping. Interestingly, T24T has acquired 4 new structural changes, 3 of which [add(10)(p12), i(10)(q10), -15] have been observed in loss of heterozygosity (LOH) studies of tumor progression in human TCC. It appears that the T24/T24T model may be an excellent tool for the study of human TCC progression because of its relationship with known karyotypic changes associated with human bladder cancer progression. We are currently taking advantage of these paired cell lines to identify genes involved in human TCC progression. Genes Chromosomes Cancer 27:252-263, 2000.

The relationship of BRMS1 and RhoGDI2 gene expression to metastatic potential in lineage related human bladder cancer cell lines.

We have recently characterized a human bladder cancer cell line T24 and a more aggressive lineage related variant of it, T24T. To gain further insights, we have studied their metastatic ability in an in vivo model system. Results show that T24 forms significantly fewer [4/12 (1/11) mice had metastases with 1-2 lesions/mouse] metastasis in SCID/bg mice than T24T [14/14 (6/6) mice had metastases with a mean of 24-28 lesions/mouse]. To begin exploring the mechanisms underlying this difference, we evaluated the mRNA and protein expression levels of metastasis-suppressor genes, known to be important in the progression of other cancers, in our model of bladder cancer progression. A higher mRNA expression of BRMS1, a metastasis suppressor in breast cancer, was observed in T24 cells. In addition, RhoGDI2 mRNA expression was only observed in T24 when compared to T24T, suggesting that Rho activation might play a significant role in the metastatic cascade. However, a basal level mRNA expression of KISS1, described as metastasis suppressor in melanoma and breast, was observed in both the lines and had slightly higher expression in T24T. No difference of Nm23-H1, KAI1, MKK4/SEK1 and E-Cadherin protein levels were noted between these two lines. In summary, it appears that the T24/T24T paired cell lines constitute a useful model for the study of human bladder cancer metastasis that will allow both the discovery and mechanistic evaluation of genes potentially involved in this process.

Prognostic markers in localized prostate cancer: from microscopes to molecules.

Management of patients diagnosed with localized prostate cancer is complicated by the diverse natural history of the disease and variable response to treatment. Prognostic criteria currently in use cannot fully predict tumor behavior and thus limit the ability to recommend treatment regimens with the assurance that they are the best course of action for each individual patient. The search for better prognostic markers is now focussed on the molecular mechanisms which underlay tumor behavior, such as altered cell cycle progression, apoptosis, neuroendocrine differentiation, and angiogenesis. As the number of potential molecular markers increases, it is becoming evident that no single marker will provide the prognostic information necessary to make a significant improvement in patient care. In addition, it seems likely that traditional methods of assessing the prognostic value of this multitude of new markers will prove inadequate. In this review, we briefly examine the current state of prognostication in localized prostate cancer and some of the promising new molecular markers. Next, we examine how new technologies may allow the multiplex analysis of vast numbers of markers and how computational methods such as artificial neural networks will provide meaningful interpretation of the data. In the near future, such an integrated approach may provide a comprehensive prognostic tool for localized prostate cancer.

Autosomal-dominant polycystic kidney disease in the rat.

Kaspareit-Rittinghausen described a rodent model of inherited polycystic kidney disease (PKD), the Han:SPRD rat [1, 2], in which heterozygotes develop renal cysts and renal failure (in males) over several months, whereas homozygous animals develop rapidly progressive renal enlargement that leads to death in a few weeks. In this study, we examined selected elements of the pathogenesis of this disease in heterozygotes and homozygotes from birth to advanced disease. Heterozygous male rats developed slowly progressive renal cystic disease with interstitial fibrosis and azotemia seen by six months of age. Female heterozygotes developed slowly progressive renal cystic disease, but did not develop interstitial fibrosis or azotemia. Epithelial cells lining cyst cavities showed various degrees of morphologic immaturity. Cyst walls also developed basement membrane thickening, especially in areas of cellular immaturity, suggesting an interrelationship between this basement membrane thickening and cellular dedifferentiation. Thickened basement membranes were associated with increased immunoreactivity for type IV collagen, laminin, and fibronectin. Homozygous rats developed massive renal enlargement, marked azotemia, and died near three weeks of age. Renal c-myc proto-oncogene expression was elevated in homozygous cystic infants and in adult heterozygotes. In situ hybridization showed high levels of c-myc mRNA in cyst epithelia, suggesting abnormal regulation of cellular proliferation in the cells lining cysts, as seen in other models of PKD. The Han:SPRD rat is the only well-documented animal model of inherited PKD with an autosomal-dominant inheritance pattern and appears to have several features which resemble human ADPKD.

Localization of overexpressed c-myc mRNA in polycystic kidneys of the cpk mouse.

The C57BL/6J-cpk mouse has a form of autosomal-recessive polycystic kidney disease characterized by the rapid growth of large collecting duct cysts and the development of severe renal failure usually by three to four weeks of age. Previous studies had shown higher steady-state levels of proto-oncogene mRNA in these cystic kidneys. It is now shown using nuclear run-on transcription that the c-fos and c-myc proto-oncogenes are transcribed at higher rates in cystic kidneys, and thus that increased transcription, in part, may account for the increased mRNA levels. c-myc mRNA was detected by in situ hybridization in nephron anlagen and elongating tubules of normal and cystic kidneys during late fetal and early neonatal kidney development. Localization of c-myc expression in the normal kidney decreased with age over the three-week postnatal period. By contrast, c-myc mRNA was found in cysts as early as three days of age, with increased levels at two and three weeks. c-myc expression was also elevated in apparently normal, non-dividing proximal tubules in three-week-old cystic animals. On the basis of these findings, we suggest that c-myc expression is linked to the proliferation of cells engaged in the primary cystogenic process, and that expression of this gene in proximal tubule cells of severely azotemic animals reflects the compensatory response of residual tubular epithelial cells to progressive renal dysfunction.

The SGP-2 gene is developmentally regulated in the mouse kidney and abnormally expressed in collecting duct cysts in polycystic kidney disease.

Sulfated glycoprotein-2 (SGP-2) is a secreted, dimeric, glycosylated protein synthesized by a number of different epithelial cell types. Although its function is not yet understood, SGP-2 has been hypothesized to be involved in such diverse processes as the promotion of cell-cell interactions, spermatogenesis, modulation of the complement system, and programmed cell death. We have now found that the SGP-2 gene is developmentally regulated in the mouse kidney. SGP-2 gene expression is first detected in the condensing nephrogenic mesenchyme and is subsequently down-regulated during the maturation of the glomerular epithelia, proximal tubules, and collecting ducts. SGP-2 continues to be expressed in the mature kidney in distal tubules and in the urothelial lining of the calyx and papilla. We have also examined the expression of the SGP-2 gene in polycystic kidneys of the C57BL/6J-cpk mouse, a model of autosomal recessive polycystic kidney disease in which there is development of epithelial-lined cysts arising primarily from the collecting duct system. Abnormally high levels of SGP-2 mRNA were found in the cyst wall epithelium of polycystic kidneys. The expression of the SGP-2 gene in normal development suggests that it plays a role in differentiating epithelial structures; and the abnormally high levels of SGP-2 gene expression in polycystic kidneys suggests that the cells lining cysts are not fully differentiated. It is possible, therefore, that polycystic kidney disease is caused by a defective developmental process in which there is a delay in terminal differentiation.

The heat shock response in HeLa cells is accompanied by elevated expression of the c-fos proto-oncogene.

Several known inducers of the heat shock response (heat stress, arsenite, and heavy metals) were shown to cause a significant elevation of c-fos mRNA in HeLa cells. Heat stress resulted in a time- and temperature-dependent prolonged elevation in the level of c-fos mRNA, which was accompanied by increased translation of c-fos protein and its appearance in the nucleus. Elevated expression of c-fos during heat stress was paralleled by induction of hsp 70 mRNA, while levels of c-myc and metallothionein mRNAs declined. Treatment of HeLa cells with arsenite or heavy metals also resulted in increased levels of hsp 70, as well as c-fos mRNA. Although elevated expression of c-fos was prevented by inhibitors of RNA synthesis, analysis of relative rates of gene transcription showed that during heat stress there was a negligible change in c-fos transcription. Therefore, the enhanced expression of c-fos during the heat shock response is likely to occur primarily through posttranscriptional processes. Cycloheximide was also shown to significantly increase the c-fos mRNA level in HeLa cells. There results are consistent with the observation that these inducers of the heat shock response, as well as cycloheximide, repress protein synthesis and suggest that the increase in the level of c-fos mRNA is caused by an inhibition of protein synthesis. This supports the hypothesis that c-fos mRNA is preferentially stabilized under conditions which induce the heat shock response, perhaps by decreased synthesis of a short-lived protein which regulates c-fos mRNA turnover.

Social implications of multiple pathology.

The degree of multiple pathology in 184 consecutive patients admitted to a geriatric unit was recorded using the 13 commonest conditions present at the time of admission to provide a standard group of disorders for comparison. All of the conditions were chronic in nature, and 35% of the patients had a combination of four or more of these present. Multiple pathology was associated with poor prognosis in terms of mortality and the need for long-term institutional care. Families, including those who were elderly, continued to support most of the frail elderly people even in the presence of multiple pathology. This emphasizes the social implications of multiple pathology and the need for support and information in the management of long-term disability.

Answers to wh-questions: a developmental study.

Forty children aged 3-6 years responded to items representing nine wh-question forms. Questions referred to three types of referential sources (conditions) based on immediacy and visual availability. The hierarchies of wh-question forms based on degree of difficulty were similar to those described in previous investigations. However, a significant interaction between referential conditions and wh-form was found to influence the relative complexity of the stimulus questions. The children were significantly less successful in giving appropriate and accurate responses when the question referred to objects, persons, or events not represented in the immediate setting. Recognition and delivery of the general category or kind of information required by a wh-form (functional appropriateness) appeared to predate substantially the ability to respond with fact, accuracy, logic, and credibility (functional accuracy). The results suggest consideration and control of referential source as well as appropriateness/accuracy response criteria in the evaluation and treatment of language-disordered children.