Mer receptor tyrosine kinase inhibition impedes glioblastoma multiforme migration and alters cellular morphology.

Glioblastoma multiforme (GBM) is an aggressive brain tumor, fatal within 1 year from diagnosis in most patients despite intensive multimodality therapy. The migratory and microscopically invasive nature of GBM as well as its resistance to chemotherapy renders conventional therapies inadequate in its treatment. Although Mer receptor tyrosine kinase (RTK) inhibition has been shown to decrease the long-term survival and improve the chemosensitivity of GBM in vitro, its role in malignant cellular migration has not been previously evaluated. In this study, we report for the first time a role for Mer RTK in brain tumor migration and show that Mer inhibition profoundly impedes GBM migration and alters cellular morphology. Our data demonstrate that Mer RTK inhibition results in altered signaling through focal adhesion kinase (FAK) and RhoA GTPase and a transformation of cytoskeletal organization, suggesting both molecular and structural mechanisms for the abrogation of migration. We also describe a novel and translational method of Mer RTK inhibition using a newly developed monoclonal antibody, providing proof of principle for future evaluation of Mer-targeted translational therapies in the treatment of GBM. Previous findings implicating Mer signaling in glioblastoma survival and chemotherapy resistance coupled with our discovery of the role of Mer RTK in GBM cellular migration support the development of novel Mer-targeted therapies for this devastating disease.

A retrospective analysis of oral hairy leukoplakia in South Australia.

BACKGROUND: The features of oral hairy leukoplakia (OHL) have been widely reported in the literature. However, no studies have described this lesion in the Australian setting. This study retrospectively examines, with respect to specific clinical factors, the prevalence of OHL in a South Australian HIV-infected population. METHODS: Clinical data were collected from the records of 197 HIV-infected patients who had attended the Adelaide Dental Hospital between January 1986 and February 1995. Data were analysed using the chi-square test. RESULTS: The prevalence of OHL in South Australian HIV-infected patients was 45.2 per cent. The study found the presence of OHL was not related to CD4+ T-lymphocyte count or AIDS-defining illness nor did the length of time a patient had been infected with HIV relate to the presence of OHL. An association was observed between a reduced prevalence of OHL in patients who were taking antiviral medication. CONCLUSION: The prevalence of OHL in South Australia is comparable with results of other studies. This study supports the notion that OHL is not an indicator of immunosuppression in South Australian HIV-infected patients. Further longitudinal studies are required to ascertain the relationship of OHL to HIV disease progression.

The localisation of ED1 antibody in the resorbing hard tissues of the periodontium.

A dental hard tissue resorptive model was used to determine the periodontal ligament (PDL) distribution of lysosomal membrane antibody ED1 to cells of the macrophage-phagocyte lineage. Immunolabel was identified in mononuclear cells around inflammatory sites in the PDL, while multinuclear cells were labelled in resorption bays present in both bone and dentine. As repair of the tissues occurred, the label became less obvious. The presence of strong ED1 label in alveolar bone marrow provided evidence supporting the haemopoietic origin of the similarly labelled PDL cells. Also, evidence confirming the current theory that multinucleated resorptive cells differentiate along a monocyte-macrophage pathway was provided. t was concluded that ED1 is a positive PDL marker for mononuclear and multinuclear cells involved in the inflammatory and resorptive processes.

Lip cancer in South Australia, 1977-1996.

Lip cancer (140 ICD-9, C00 ICD-10) is a form of oral cancer occurring at the junction between the oral cavity and the skin. Lip cancer has a distinctive global epidemiology that is notably different from cancer occurring at other intraoral sites. This study reviews and analyses the epidemiological data for lip cancer from the South Australian Central Cancer Registry between 1977 and 1996. During this 20-year period, 2716 cases of lip cancer (2095 male, 621 female) and 35 deaths from this disease (23 males, 12 females) were reported. The average age of diagnosis was 58.3 years in males and 66.0 years in females. Very high age-standardised incidence rates (over 15.0 per 100000 per annum in males and 4.0 per 100000 per annum in females) were found, giving the South Australian population amongst the highest incidence of lip cancer in the world. Also of considerable concern was the finding that, contrary to global trends, these rates showed a significant increase over the 20-year period in both sexes. Possible reasons for these findings are discussed.

Reciprocal activation of xenobiotic response genes by nuclear receptors SXR/PXR and CAR.

The cytochrome P450 (CYP) gene products such as CYP3A and CYP2B are essential for the metabolism of steroid hormones and xenochemicals including prescription drugs. Nuclear receptor SXR/PXR (steroid and xenobiotic receptor/pregnenolone X receptor) has been shown both biochemically and genetically to activate CYP3A genes, while similar studies have established constitutive androstane receptor (CAR) as a CYP2B regulator. The response elements in these genes are also distinct, furthering the concept of independent regulation. Unexpectedly, we found that SXR can regulate CYP2B, both in cultured cells and in transgenic mice via adaptive recognition of the phenobarbital response element (PBRE). In a type of functional symmetry, orphan receptor CAR was also found to activate CYP3A through previously defined SXR/PXR response elements. These observations not only provide a rational explanation for the activation of multiple CYP gene classes by certain xenobiotics, but also reveal the existence of a metabolic safety net that confers a second layer of protection to the harmful effects of toxic compounds and at the same time increases the propensity for drug-drug interactions.

The epidemiology of mouth cancer: a review of global incidence.

Mouth cancer (143-145 ICD-9) is a major health problem in many parts of the world. While its incidence is relatively low in most western countries there are some important exceptions to this trend: on the Indian subcontinent and in other parts of Asia it remains one of the most common forms of cancer. This review article summarises the global incidence of mouth cancer using cancer maps. Data have been compiled from the latest edition of Cancer Incidence in Five Continents and recent studies from various locations around the world. Significant geographic variation is noted in the incidence of mouth cancer, with high rates reported for the Indian subcontinent and parts of Asia (male incidence rates in excess of 10 per 100,000 per annum). It is also noted that as with other forms of oral cancer, the majority of population-based data for mouth cancer comes from the Western world with a paucity of reliable data from the so-called developing countries. Mouth cancer remains a serious health problem in many parts of the world with many regions reporting increasing incidence rates particularly in males. Ongoing research into the aetiologic risk factors associated with this disease must remain a very high priority if the causes of mouth cancer are to be established and disease control protocols introduced widely.

The epidemiology of tongue cancer: a review of global incidence.

The tongue (141 ICD-9) is the most common intraoral site for cancer in most countries, however its global epidemiology shows significant geographic variation. This review paper summarises the global incidence of cancer of the tongue using cancer maps and references to recent studies from various locations. Tongue cancer remains a serious health problem in many countries including India (male incidence rates up to 6.5 per 100,000 per annum) and parts of Europe (male incidence rates in France up to 8.0 per 100,000 per annum). It is noted that as with other forms of oral cancer the majority of population-based data for tongue cancer comes from the Western world with a paucity of reliable data from the so-called developing countries. The tongue remains the most common intraoral site for oral cancer worldwide and in a number of countries it is a serious public health problem with significant morbidity and mortality. While the incidence of tongue cancer appears to be stable or falling in some regions of the world, in other areas it is rising, particularly among younger people.

Hypothermic insult to the periodontium: a model for the study of aseptic tooth resorption.

The aim of the current investigation was to define an animal model for the study of hard tissue resorption by examining the responses of the periodontal ligament (PDL) to both single and multiple episodes of hypothermic injury to the crowns of rat teeth. A group of 12 male rats weighing 200-250 g were anesthetized, and pellets of dry ice (CO2) were applied once to the crowns of the right first maxillary molars for continuous periods of 10 or 20 min. Animals were sacrificed at 2, 7, 14 and 28 days and tissues were processed for routine histological examination. A second group of eight animals and a third group of 12 animals were subjected to three applications of dry ice over a period of 1 week and sacrificed at 2 and 14 days respectively after the final application. In addition to thermal insult, the periodontium of teeth from a fourth group of six rats was subjected to mechanical trauma. Examination of the sections from the group undergoing a single freezing episode revealed that, by 1 week, shallow resorption lacunae had appeared on the root surface. These became more extensive after 14 days. At the same time hyaline degeneration was evident in the PDL. Within this group, teeth subjected to the longer 20-min application times generally showed more extensive injuries. By 28 days, evidence of repair was observed with reparative cementum beginning to line the resorption lacunae in the root dentin. Sections from animals subjected to multiple episodes of thermal trauma and those subjected to additional mechanical insult showed more extensive external root resorption than those from single-injury animals. It was concluded that low temperature stimuli applied to the crowns of rat molars were capable of eliciting a sterile degenerative response in the PDL which, in turn, resulted in external root resorption. Furthermore, the degree of this tissue injury was commensurate with the duration and number of exposures to the trauma. The results also indicated that progression of the resorptive process required periodic exposure to the injury, in the absence of which repair to the damaged root occurred.

Oral cancer: role of the basement membrane in invasion.

Invasive growth of cancer cells is a complex process involving specific interactions between tumour cells and the orderly, integrated complexes of the extracellular matrix. Basement membranes have been proposed as one constituent of extracellular matrix which carries responsibility for regulating invasion and metastasis. Using a chemically induced rat tongue carcinoma model, it has been shown that components of the basement membrane and its overall structure are altered during tumour invasion, and methods have been developed to quantitate some of these differences. Since the basement membrane can be specifically characterized by its fibrous protein network of Type IV collagen and laminin, which is embedded in a heparan sulphate-rich proteoglycan matrix, these components have been targeted. In particular, the current paper presents results in the context of current concepts of early changes in neoplastic invasion of underlying connective tissues. In consequence, further elaboration of the underlying mechanisms of epithelial migration in oral cancer may allow an exploration of the use of alterations in expression of basement membrane components as prognostic indicators.

E2F1 has both oncogenic and tumor-suppressive properties in a transgenic model.

Using a transgenic mouse model expressing the E2F1 gene under the control of a keratin 5 (K5) promoter, we previously demonstrated that increased E2F1 activity can promote tumorigenesis by cooperating with either a v-Ha-ras transgene to induce benign skin papillomas or p53 deficiency to induce spontaneous skin carcinomas. We now report that as K5 E2F1 transgenic mice age, they are predisposed to develop spontaneous tumors in a variety of K5-expressing tissues, including the skin, vagina, forestomach, and odontogenic epithelium. On the other hand, K5 E2F1 transgenic mice are found to be resistant to skin tumor development following a two-stage carcinogenesis protocol. Additional experiments suggest that this tumor-suppressive effect of E2F1 occurs at the promotion stage and may involve the induction of apoptosis. These findings demonstrate that increased E2F1 activity can either promote or inhibit tumorigenesis, dependent upon the experimental context.

Chlorhexidine as a root canal medicament for treating inflammatory lesions in the periodontal space.

The purpose of the present study was to investigate the therapeutic effect of intra-canal application of chlorhexidine on inflammatory root resorption. Dental pulps from monkey incisors were infected and resealed prior to extraction of the teeth. Root dentin was mechanically exposed and the teeth were replanted under aseptic conditions. After 4 weeks, the experimental teeth were dressed with chlorhexidine gel and resealed. Animals were sacrificed 4 weeks later, and their jaws prepared for histologic examination. Results showed that both marginal and apical periodontal inflammation and resorption were reduced in the chlorhexidine-treated teeth. It was concluded that the use of intra-canal chlorhexidine may be a useful adjunct in the treatment of inflammatory root resorption, but further human trials need to be undertaken before its clinical use can be recommended.

Differential activities of E2F family members: unique functions in regulating transcription.

Several regulators of E2F transcriptional activity, including the retinoblastoma tumor suppressor (Rb) protein, p16Ink4a, cyclin D1, and cyclin-dependent kinase 4, have been shown to be targets for genetic alterations that underlie the development of human cancers. Deregulation of E2F transcription factors as a result of these genetic alterations is believed to contribute to tumor development. This hypothesis is supported by the finding that at least some members of the E2F gene family can contribute to oncogenic transformation when overexpressed. Each E2F family member can dimerize with DP proteins, bind consensus E2F sites, and activate transcription. Several pieces of evidence suggest, however, that the various E2F species have unique functions in regulating transcription. We compared the abilities of E2F1, E2F4, and E2F5 to activate transcription from a variety of gene promoters and found that in all cases E2F1 was the most potent activator, followed by E2F4 and then by E2F5. Construction of chimeric proteins between E2F1 and E2F4 demonstrated that either the carboxy terminus or the amino terminus of E2F1 could make E2F4 a more potent activator. In contrast, neither the carboxy terminus nor the amino terminus of E2F1 could significantly increase the activity of E2F5. We found that, consistent with a role for E2F5 in transcriptional repression, E2F5's binding partner p130, like Rb, could also actively repress transcription when directly bound to a target promoter.

Increased E2F1 activity induces skin tumors in mice heterozygous and nullizygous for p53.

The p16(INK4a)-cyclin D-retinoblastoma tumor suppressor pathway is disrupted in most human cancers, and it has been suggested that the subsequent release of E2F transcription factors from inhibitory complexes may be a key event in tumor development. We described recently the generation of transgenic mice with E2F1 gene expression targeted to squamous epithelial tissues by a keratin 5 (K5) promoter. In the present study, K5 E2F1 transgenic mice were crossed with p53 null mice to examine functional interactions between E2F1 and p53 in vivo. We find that E2F1-induced apoptosis of epidermal keratinocytes is reduced in K5 E2F1 transgenic mice lacking p53, whereas E2F1-induced hyperproliferation is unaffected by p53 status. We also find that K5 E2F1 transgenic mice heterozygous or nullizygous for p53 develop spontaneous skin carcinomas, which normally are rare in p53-deficient mice. The timing of tumor development correlates with the level of E2F1 transgene expression and the status of p53. In primary transgenic keratinocytes, the major change in E2F1 DNA-binding activity is the generation of a complex also containing the retinoblastoma tumor suppressor protein. Nevertheless, the expression and associated kinase activity of cyclin E, a known target for E2F transcriptional activity, is elevated significantly in K5 E2F1 transgenic keratinocytes. These findings firmly establish that increased E2F1 expression can contribute to tumor development and suggest that p53 plays an important role in eliminating cells with deregulated E2F1 activity.

Deregulated expression of E2F1 induces hyperplasia and cooperates with ras in skin tumor development.

In cell culture studies, overexpression of the E2F1 transcription factor has been shown to stimulate proliferation, induce apoptosis, and cooperate with an activated ras gene to oncogenically transform primary rodent cells. To study the effect of increased E2F1 activity on epithelial growth and tumorigenesis in vivo, transgenic mice expressing E2F1 under the control of a keratin 5 (K5) promoter were generated. Expression of E2F1 in the epidermis results in hyperplasia but does not inhibit terminal differentiation. In a transgenic line expressing high levels of E2F1, mice have decreased hair growth likely as a result of aberrant apoptosis in developing hair follicles. Coexpression of a cyclin D1 transgene with E2F1 augments epidermal hyperplasia and further disrupts hair follicle development suggesting that hypophosphorylated Rb antagonizes the proliferative and apoptotic-promoting activities of E2F1. Finally, the E2F1 transgene is found to cooperate with a v-Ha-ras transgene to induce skin tumors in double transgenic animals. These findings confirm that many of the activities ascribed to E2F1 through in vitro studies can be reproduced in vivo and demonstrate for the first time that deregulated E2F activity can contribute to tumor development.

Stereochemistry of yeast delta 24-sterol methyl transferase.

S-Adenosyl-l-methionine: delta 24-sterol methyl transferase (24-SMT) mediates introduction of the C-28 carbon of yeast sterols. It has been shown that sulfonium analogues of the presumptive cationic intermediates of the methylenation reaction are potent in vivo and in vitro inhibitors of this process. In the presence of these inhibitors, cultures of yeast produced increased proportions of zymosterol, the natural substrate of the enzyme, while proportions of ergosterol and ergostatetraenol were decreased. New C27-sterol metabolites were also found. The in vivo inhibitory power of the analogues [I50 (microM)] was determined from the proportion of C-24 methylated sterols to C-24 nonmethylated sterols in treated cultures to be in the following order: 25-thiacholesterol iodide (0.07) > 24(S)-methyl-25-thiacholesteryl iodide (0.14) > 24(R)-methyl-25-thiacholesteryl iodide (0.25). Kinetic inhibition as revealed by radiolabeled S-adenosyl-l-methionine (SAM), crude enzyme and 25-thiacholesteryl iodide revealed this inhibitor to be uncompetitive with respect to zymosterol and competitive with respect to SAM. The greater inhibitory power of 24(S)-methyl-25-thiacholesteryl iodide compared to 24(R)-methyl-25-thiacholesteryl iodide suggests that methyl donation to delta 24 occurs from the si face. When considered in conjunction with Arigoni's previous work, the present results infer the methylenation mediated by yeast 24-SMT proceeds by alkylation from the si face of delta 24 followed by migration of a hydrogen from C-24 to C-25 across the re face and final loss of a hydrogen from C-28 on the re face.

Therapeutic delivery of calcitonin to inhibit external inflammatory root resorption. I. Diffusion kinetics of calcitonin through the dental root.

Insertion of calcitonin into root canals of monkey teeth has been shown to inhibit external inflammatory root resorption and suppress inflammation. Regulation of this therapeutic event depends upon the rate of arrival (diffusion) of the hormone at sites of resorptive activity. In the present study, the diffusion characteristics of calcitonin through the dental root in an extracted human-tooth model are described, and the role of cementum in the diffusion process is also addressed. Root-canals were endodontically prepared to form a reservoir for [125I]-calcitonin, and macerated to remove organic material from dentinal tubules. In teeth with intact cementum, an initial period of delay (4-5 h) prior to the detection of calcitonin at the external tooth-root surface was followed by a rapid release of the calcitonin during the first 10.5 h (rate peaks at 6 h). Slower, sustained releases of calcitonin through intact cementum were measured for the following 9 days. Removal of cementum, to expose "smear-free" dentine, resulted in an earlier efflux of calcitonin (2 h) at external tooth surfaces and increased amounts of calcitonin release over 9 days. Biphasic delivery of calcitonin by such internal diffusion mechanisms suggests that loss of cementum will enhance therapeutic availability, while prolonged delivery to intact external dental-root surfaces following early intra-canal placement may also be useful for the therapeutic prevention of external inflammatory root resorption.

Therapeutic delivery of calcitonin to inhibit external inflammatory root resorption. II. Influence of calcitonin binding to root mineral.

Experimentally-induced external inflammatory tooth-root resorption can be inhibited by therapeutic doses of calcitonin. Such doses can be delivered by an intrinsically slow diffusion pathway, from a reservoir in endodontically-debrided root canals, via the dentinal tubules. While the kinetics of this journey have been followed in an earlier report, the binding characteristics of calcitonin to the tooth mineral, which will be responsible, in part, for these kinetics, have not been reported before. The current study examines the binding potential of calcitonin to root mineral and addresses the potential role of non-specific binding proteins. A modified Scatchard plot indicated that a simple non-reactive type of ligand binding exists between calcitonin and root mineral, represented by a small number of identical binding sites. This interaction is both strong and reversible. Furthermore, it appears to be time-dependent with more time being required for the residual ligands to interact with the diminishing numbers of free calcitonin-binding sites. While preloaded [125I]-calcitonin could be incompletely (75-91%) displaced from dental-root material by non-radioactive calcitonin, its release was slow over 23 h. Calcitonin was four times as effective as bovine-serum albumin in competing for common "calcitonin binding sites" on macerated dental-root material. Thus, even in the presence of extraneous protein, calcitonin will bind tightly but reversibly to tooth-root material, making it a good candidate for therapeutically protracted delivery to external root surfaces from root canals.

Palatal changes associated with reverse smoking in Filipino women.

OBJECTIVE: To determine baseline data for the presence or absence of reverse-smoking and conventional smoking associated oral palatal mucosal changes in women. DESIGN: A cross-sectional evaluation of the clinical and cytological changes associated with the condition. SETTING: Nine rural barangays in Cabanatuan City, Philippines. SUBJECTS AND METHODS: Nine-one volunteer women smokers (61 reverse and 30 conventional) were examined clinically and photographically. Smears were also taken from three areas and the palate to investigate the cytology of palatal mucosal epithelium. MAIN OUTCOME MEASURES: Variations in colour, texture and topography of the palatal mucosa; determination of epithelial cell characteristics and inflammatory cell populations present in the lesions. RESULTS: Clinical findings showed that subjects could be grouped into three categories: Group A subjects showed pigmentation and some erythema only; Group B subjects included those with ulceration, marked erythema and non-descript mucosal roughening; Group C subjects (comprising the majority of reverse smokers) exhibited various combination of leukoplakia, fissuring, thickening and pigmentation of the palatal mucosa. Additional features, including nodularity, erythema, prominence and reddening of minor salivary gland duct openings were also occasionally observed in this group. Cytologic analysis revealed that, within each smoking group, there was a significant site-dependent difference in the predominant epithelial cell type present. CONCLUSIONS: This study reports the first systematic description of reverse smoking associated palatal mucosal changes in Filipino women. It also provides a basis for classification of the palatal mucosal changes among reverse smokers.