Comparative achievement on National Dental Board Examination Part I between dental students in problem-based learning and traditional educational tracks.
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California↗
Biomedical subjects
C F Shuler
Publications and source records attributed to C F Shuler.
Transforming growth factor-beta3 regulates transdifferentiation of medial edge epithelium during palatal fusion and associated degradation of the basement membrane.
Studies on transforming growth factor beta3 (TGF-beta3) deficient mice have shown that TGF-beta3 plays a critical role in palatogenesis. These null mutant mice have clefting of the secondary palate, caused by a defect in the process of fusion of the palatal shelves. A critical step in mammalian palatal fusion is removal of the medial edge epithelial cells from the midline seam and formation of continuous mesenchyme. To determine in more detail the role of TGF-beta3 in palatogenesis, we cultured TGF-beta3 null mutant and wild-type control palatal shelves in an organ culture system. The fate of the medial edge epithelial cells was studied in vitro using vital cell labeling and immunohistochemical techniques. Despite clear adherence, the null mutant palatal shelves did not fuse in vitro, but instead the medial edge epithelial cells survived at the midline position, and the basement membrane was resistant towards degradation. Supplementation of the culture medium with the mature form of TGF-beta3 was able to fully correct the defective fusion in the null mutant specimens. Our results demonstrate that the reason for the defective palatal fusion in TGF-beta3 (-/-) samples is not impaired adhesion. Our data define a specific role for TGF-beta3 in the events that control transdifferentiation of the medial edge epithelial cells including degradation of the underlying basement membrane.
Animals↗
Ineffectiveness of the presence of H-ras/p53 combination of mutations in squamous cell carcinoma cells to induce a conversion of a nontumorigenic to a tumorigenic phenotype.
Human tumor cells have properties in vitro or in surrogate hosts that are distinct from those of normal cells, such as immortality, anchorage independence, and tumor formation in nude mice. However, different cells from individual tumors may exhibit some, but not all of these features. In previous years, human tumor cell lines derived from different tumor and tissue types have been studied to determine those molecular changes that are associated with the in vitro properties listed above and with tumorigenicity in nude mice. In the present study, seven cell lines derived from human tumors were characterized for p53 and ras mutations that may occur in SCC tumor phenotypes and for tumor formation in nude mice. This investigation was designed to examine whether co-occurrence of mutated ras and p53 lead to a malignant stage in the progression process. None of the seven cell lines contained mutations in the recognized "hot spots" of the p53 tumor suppressor gene, but four had a nonsense/splice mutation in codon 126 and a mutation in codon 12 of the H-ras gene. The remaining three cell lines had p53 mutations in intron 5, in codon 193, and a missense mutation in codon 126, respectively. Four of seven cell lines were nontumorigenic; two of these cell lines contained a nonsense p53-126 mutation and mutated ras; one had a missense mutation at codon 126 but no mutated ras; the the fourth had only a p53 mutation at codon 193. Two of the nontumorigenic cell lines were converted to tumorigenicity after treatment with methyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine with no apparent additional mutations in either gene. Our analysis revealed that there was a high frequency of genetic diversity and mutations in both p53 and H-ras. There was also a lack of a causal relationship in the presence of mutations in p53 and the cells' ability to exhibit a malignant potential in nude mice.
Animals↗
Problem-based learning at the University of Southern California School of Dentistry.
Responding to the recent Institute of Medicine report on dental education, the Center for Craniofacial Molecular Biology (CCMB) of the University of Southern California School of Dentistry has developed a parallel track program in dental education leading to the D.D.S. degree. This program was proposed in May of 1995, and the first class of twelve students was admitted in September of that year. Currently two classes are enrolled and plans to admit a further twelve students (Class of 2001) are in place. The educational strategy for this program is totally problem-based. Students work in groups of six with a faculty facilitator, not necessarily a content expert. Facilitators are largely drawn from the multidisciplinary pool of research faculty at the center. All learning is mediated through biomedical and biodental problem cases. No formal lectures or classes are scheduled. The learning of clinical dental skills is promoted through focussed dental patient simulations in which students review clinical charts, radiographs, medical reports and then explore identified, hands-on learning needs using patient simulators in a clinical context. Early patient exposure is obtained through dental office visits and other special patient clinics. Initial experience with this program suggests that the problem-based learning (PBL) students learn as well (if not better) than their traditional program peers and develop excellent group and cognitive analytical skills. The absence of a pool of dentally related biomedical cases suitable for a PBL program has necessitated the use of innovative approaches to their development and presentation. It is believed that this educational approach will produce dental clinicians equipped with the self-motivated, life-long learning skills required in the ever-changing world of bio-dental sciences in the twenty-first century.
California↗
Xenograft growth and histodifferentiation of squamous cell carcinomas of the pharynx and larynx.
The potential for growth in a xenogeneic host and the pattern of histodifferentiation was examined in 56 human squamous cell carcinomas of the pharynx and larynx transplanted to nude mice. Among these 56 transplanted tumors, 13 of 36 primary and 12 of 20 metastatic tumors were found to be tumorigenic in nude mice. Eleven of 24 pharyngeal and 14 of 32 laryngeal tumors grew in nude mice. The dynamics of transplanted tumor growth in the surrogate host took place independent of the tumor grade and site of tumor origin. Transplanted tumors exhibited a higher mitotic index compared with the original tumors. Each passage of a tumor line resulted in most of the xenograft tumors exhibiting an increased degree of differentiation without invasion of host tissues. No difference was observed between patients who engrafted tumors in the xenograft model and those who did not with respect to tumor biology or clinical findings, including survival.
Animals↗
A conundrum in molecular toxicology: molecular and biological changes during neoplastic transformation of human cells.
The process of multistage carcinogenesis lends itself to the concept that the effects of carcinogens are mediated through dose-related, multi-hit, linear changes. Multiple in vitro model systems have been developed that are designed to examine the cellular changes associated with the progression of cells through the different stages in the process; however, these systems may have inherent limitations due to the cell lines used for these studies, the manner of assessing the effects of the carcinogens, and the subsequent growth and differentiation of the exposed cells. Each of these variables results in increasing levels of uncertainty relative to the correlation of the events with the actual process of human tumor development. Therefore, the prediction of the ultimate effect of any carcinogen is difficult. Moreover, relationships between individual biological endpoints resulting from carcinogen treatment appear at best to be approximations. The presence of an activated carcinogen inside the cell can give rise to multiple outcomes, only some of which may be critical events. For example, site-specific modification of the 12th and 13th codons of H-ras is different than that in the adjacent 14th and 15th codons. It is interesting to speculate what effect these differences might have on a biological outcome, e.g., transformation to anchorage-independent growth. The use of different model systems to examine the effects of activated carcinogens also creates additional problems. Comparisons of in vitro transformed cells with similar cells isolated from human tumors indicate that the culture environment appears to influence the expression of a particular phenotype, in that human tumor cells in culture express many of the same parameters as those found in cells transformed with carcinogens in vitro. If the process of transformation is linear, then less aggressive phenotypes should progress to a more aggressive transformed stage. However, in carcinogen-transformed human cells, the populations exhibit phenotypic diversity in that many of the transformed cells differentiate and fail to continue to divide in culture. Historically, we have assumed only a limited role for epigenetic modulation of molecular changes that occur during progression; however, our data suggest quite strongly that nonmalignant tumor populations can be converted to a more malignant phenotype without additional mutations taking place and, conversely, malignant populations can be downregulated to a nontumorigenic phenotype. Tumor cell plasticity is not only a fundamental characteristic of diverse types of human tumors, but also appears as an integral characteristic of carcinogen-transformed cells in vitro.
Base Sequence↗
Mesenchymal commitment to digital joint formation.
Temporal and spatial commitment of in vivo and in vitro mammalian digital joint development were characterized in a murine model. Alcian blue and alizarin red staining were used to label proteoglycans of cartilage matrix and mineralized matrix in both whole mounts and histological sections. Mesenchymal differentiation toward a joint fate was identified by a lack of matrix deposition in islands of joint precursor cells between phalangeal precursors, and localized lysosomal enzyme activity was later demonstrated in these regions during formation of the joint cavity. Organ-cultured forelimbs and in vivo specimens demonstrated analogous digital joint morphological trends. With a defined developmental window, reverse transcription, polymerase chain reaction, demonstrated differential gene expression of transforming growth factor-beta isotypes, aggrecan core protein, and type II collagen, suggesting a role for transforming growth factor-beta in directing digital joint development.
Actins↗
In vitro model of syndactyly replicates the morphologic features observed in vivo.
Syndactyly is a common congenital hand anomaly that may occur after exposure to teratogens. We have developed an in vitro model of syndactyly to investigate the molecular mechanisms underlying this malformation of digit development. Retinoic acid, which regulates pattern formation in vertebrate limb development and is associated with teratogenic malformations, was used in the development of this syndactyly model system. Pregnant Swiss-Webster mice were given retinoic acid by oral gavage on days 10 and 11 of embryonic development (E10 and E11, respectively). The mice were sacrificed on gestational days 13 and 17 (E13, E17) and immediately postnatally (PN). The fetuses were removed and the forelimbs dissected under the operating microscope. The E13 limbs were cultured for 4 days (E13+4) in an organ culture system using a serumless, chemically defined medium. The E17, PN, and E13+4 forelimbs were critically examined for malformations of digit separation and digit development. Retinoic acid-induced fetal mouse forelimb syndactyly was observed in all the groups; 81 percent of E17 limbs, 75 percent of PN limbs, and 77 percent of E13+4 limbs had syndactyly. The morphology of the digital malformations was similar in the E17, PN, and E13+4 limbs. This in vitro model permits further studies to characterize the molecular changes that occur during the development of a congenital hand anomaly.
Animals↗
Programmed cell death and cell transformation in craniofacial development.
Fusion of branchial arch derivatives is an essential component in the development of craniofacial structures. Bilaterally symmetric branchial arch processes fuse in the midline to form the mandible, lips, and palate. The mechanism for fusion requires several different morphologic and molecular events prior to the completion of the mesenchymal continuity between opposing tissue processes. The ectodermal covering of the branchial arches is one of the cell types that has an important role during craniofacial development. The surface epithelia provide the initial adherence between the processes; however, this population of cells is ultimately absent from the fusion zone. The medial edge epithelium of the secondary palatal shelves is one example of such an epithelium that must disappear from the fusion zone of the secondary palate during development in order to complete palatal fusion. The mechanisms for removal of the epithelial cells from the fusion zone could include either programmed cell death, epithelial-mesenchymal transformation, or migration to adjacent epithelia. All three of these fates have been hypothesized as a mechanism for the removal of the palatal medial edge epithelia. The processes of programmed cell death, epithelial-mesenchymal transformation, and epithelial migration are reviewed with respect to both palatal fusion and results reported in other model systems.
Apoptosis↗
Medial edge epithelium fate traced by cell lineage analysis during epithelial-mesenchymal transformation in vivo.
Vital cell labeling techniques were used to trace the fate of the medial edge epithelial (MEE) cells during palatal fusion in vivo. Mouse palatal tissues were labeled in utero with DiI. The fetuses continued to develop in utero and tissues of the secondary palate were examined at several later stages of palatal ontogeny. The presence and distribution of DiI was correlated with the presence of cell phenotype-specific markers. During the initial stages of palatal fusion the DiI-labeled MEE were present in the midline position. These cells were attached to an intact laminin-containing basement membrane and contained keratin intermediate filaments. At later stages of palatogenesis the DiI-labeled MEE were not separated from the mesenchyme by an intact basement membrane and did not contain keratin. In late fetal development, DiI-labeled cells without an epithelial morphology were present in the mesenchyme. The transition of the DiI-labeled cells from an epithelial phenotype to a mesenchymal phenotype is consistent with a fate of epithelial-mesenchymal transformation rather than programmed cell death.
Animals↗
Histopathologic comparison between human oral squamous cell carcinomas and their xenografts in nude mice.
Tumor portions were obtained from 11 oral squamous cell carcinomas and used to produce tumors in a xenogeneic gnotobiotic nude mouse model system. The tumors that developed were examined histologically. The cellular features and tumor growth patterns were compared between the original oral squamous cell carcinoma (T0) and the tumor that developed in the xenogeneic host (T1). The T1 tumors were more highly differentiated in comparison with the T0 tumors. The T1 tumors exhibited less cellular pleomorphism and a more uniform pattern of stratification. The cells in the T1 tumors exhibited an increased mitotic index but did not contain abnormal mitoses. The T1 tumors usually exhibited nodular growth patterns rather than the infiltrating pattern exhibited by the parenchyma of the T0 tumors. The desmoplastic reaction was markedly reduced in the T1 tumors. The results indicated that the gnotobiotic nude mouse model system either selects for or induces a more differentiated pattern of tumor growth than that found in the original human oral squamous cell carcinomas.
Adult↗
Ultrastructural characteristics of a cell line derived from a melanotic neuroectodermal tumor of infancy.
Thin section and freeze-fracture transmission electron microscopy were used to examine and identify the cytoplasmic and membrane structures in a cell line derived from a melanotic neuroectodermal tumor of infancy (MNTI). The cultured cells had a uniform appearance after 70 population doublings characterized by long dendritic processes and evidence of melanin production. The cytoplasm contained numerous melanosomes in various stages of development, vesiculated rough endoplasmic reticulum, microfilaments and uncoated as well as coated vesicles. The membrane specializations included caveoli, coated pits, gap junctions, microfilaments, desmosome-like structures and lamellipodia. The ultrastructural appearance of the cultured MNTI cells was similar to features previously seen in electron micrographs of MNTI tumor specimens. However, correlated freeze-fracture and thin section micrographs permitted further identification of structures previously described. The MNTI cell line represents one of the cell types of the tumor and provides an opportunity for further study of the pathogenesis of this rare tumor.
Cell Line↗
Molecular and morphologic changes during the epithelial-mesenchymal transformation of palatal shelf medial edge epithelium in vitro.
The fate of the medial edge epithelial (MEE) cells during palatal fusion has been proposed to be either programmed cell death or epithelial-mesenchymal transformation. Vital cell labeling techniques were used to mark the MEE and observe their fate during palatal fusion in vitro. Fetal mouse palatal shelves were labeled with Dil and allowed to proceed through fusion while maintained in an organ culture system. The tissues were examined at several stages of palatal fusion for the distribution of Dil, presence of specific antigens and ultrastructural appearance of the cells. The MEE labeled with Dil occupied a midline position at all stages of palatal fusion. Initially the cells had keratin intermediate filaments and were separated from the underlying mesenchyme by an intact basement membrane. During the process of fusion the basement membrane was degraded and the Dil-labeled MEE were in contact with the mesenchymal-derived extracellular matrix. In the late stages of fusion the Dil-labeled MEE altered their cellular morphology, had vimentin intermediate filaments, and were not associated with an identifiable basement membrane. Dil-labeled cells, without an epithelial phenotype, remained present in the midline of the completely fused palate. The data indicate that the MEE did not die but underwent a phenotypic transformation to viable mesenchymal cell types, which were retained in the palatal mesenchyme.
Animals↗
Intraoral grafting of a canine full-thickness oral mucosal equivalent produced in vitro.
A canine full-thickness oral mucosal equivalent (FTOME) was produced in vitro and used in intraoral grafting. The FTOME consisted of an autologous stratified epithelial surface layer overlying an acellular bilaminar collagenous matrix. The material was grafted on the periosteum of deepithelialized edentulous alveolar ridges in dogs. Biopsies of the grafted FTOME showed that the epithelium remained viable, migrated over the surrounding tissue, and aided in wound healing.
Alveolar Process↗
Development of neoplastic phenotype following transfection of HNF cells with sarcoma DNA.
DNA isolated from chondrosarcoma cells effectively transformed NIH-3T3 cells and human foreskin fibroblasts. The transfected NIH-3T3 cells, directly implanted three or four passages later, formed progressively growing tumors (greater than or equal to 2.0 cm in diameter) subcutaneously in nude mice. No metastasis was evident upon pathological examination of the tumor bearing mice. Transfected human foreskin fibroblasts that exhibited anchorage independent growth formed only small tumors in nude mice (less than 0.6 cm in diameter). The transfected human cells which exhibited anchorage independent growth reacted with the monoclonal antibody 345.134S, specific for an epitope expressed by human sarcoma cells. The transfected NIH-3T3 cells did not exhibit reactivity with the same monoclonal antibody. Southern blot analysis of the DNA prepared from the transfected NIH-3T3 cells, that developed as a progressively growing tumor in a nude mouse, revealed the presence of human repetitive DNA sequences.
Animals↗
Localization of H-ras mRNA in oral squamous cell carcinomas.
Expression of the H-ras oncogene was examined in 5 SCC of the oral cavity. The presence of oncogene mRNA was detected and localized in the tumor tissue by in situ hybridization. Tumor tissues were also examined for the presence of the oncogene product, p21, by immunohistochemical techniques. H-ras mRNA and p21 were detected in all 5 tumors in cells that were also positive for both keratin mRNA and keratin protein. The distribution of the mRNA was not uniform throughout the tumor tissue. Distinct spatial localizations of the H-ras mRNA were present in regions of the tumors with both higher proliferative and invasive potential. The results suggest that the distribution of the oncogene mRNA may be related to the pattern of development and progression of oral SCC.
Aged↗
Effect of periodontal dressings on human gingiva fibroblasts in vitro.
In vitro cytotoxicity studies of periodontal dressings have not generally produced a result consistent with in vivo observations. These prior in vitro studies have not used human intraoral cell lines. We tested the effects of two eugenol containing and two non-eugenol periodontal dressings on cultured human gingival fibroblasts (HGF) (ATCC #1292). Replicate HGF cultures grown in microtiter plates were exposed to stock, 1:4 and 1:16 dilutions of extracts made from each of the four periodontal dressings. The HGF cultures were pulse labelled with tritiated thymidine (3HTdR) after 24, 48, and 72 hours. Incorporations of the labelled thymidine were measured using liquid scintillation counting and expressed as counts per minute. The results showed that undiluted extracts from all four periodontal dressings totally inhibited 3HTdR uptake (P less than 0.05). The 1:4 dilution of eugenol dressings inhibited 3HTdR uptake significantly more than non-eugenol dressings (P less than 0.05). Interestingly, at 72 hours the 1:16 dilution of the non-eugenol dressings caused significantly increased 3HTdR uptake which was not observed with the eugenol dressings. The present results suggest that the use of a human fibroblastic cell line for testing the effects of periodontal dressings may provide information about the relative biological effects of these dressings. Using this cell line, we have found that eugenol dressings inhibit fibroblast proliferation to a greater extent than non-eugenol dressings.
Cell Line↗
Transformation of normal homologous cells by a spontaneously activated Ha-ras oncogene.
Several tumor-derived oncogenes have been shown to independently act as complete carcinogens following transfection into target cells from established tissue culture lines. However, the number and types of oncogenes required to transform primary cultures of normal mammalian cells is unclear. To clarify this issue in a simplified model system, we transfected genomic DNA from a naturally occurring rat tumor into NIH/3T3 cells as well as into early passage rat embryo fibroblasts. The 3T3 cells were transformed with high efficiency to malignant phenotypes; the rat embryo cells were transformed at lower frequencies following cotransfection with a selectable neomycin resistance marker and treatment with Geneticin (G418). The transformed rat cells had cancerous phenotypes as determined by in vitro, cytogenetic, and in vivo criteria. Moreover, the transformed mouse and rat cells contained new tumor DNA-derived nucleotide sequences homologous to the activated human Ha-ras oncogene. Elevated levels of Ha-ras-specific mRNA, as well as enhanced expression of the Mr 21,000 oncogene product, were detected in the transformed cells. Therefore, under well-defined experimental conditions, a spontaneously activated Ha-ras oncogene from a naturally occurring tumor was able to independently transform normal, homologous cells to a malignant phenotype.
Animals↗