Carnell Newkirk

p53 Point mutations in dysplastic and cancerous ulcerative colitis lesions

BACKGROUND The molecular basis of colorectal dysplasia and carcinoma arising in ulcerative colitis is poorly understood. Loss of heterozygosity involving the tumor suppressor gene p53 occurs frequently in neoplastic ulcerative colitis lesions. Point mutation affecting p53 is associated with loss of heterozygosity in other cancers. Therefore, it was determined whether p53 point mutation occurs in ulcerative colitis-associated neoplasia. METHODS Single-strand conformation polymorphism analysis, DNA sequencing, and loss of heterozygosity studies were performed on 45 patients with ulcerative colitis-associated dysplasia and carcinoma. RESULTS Point mutations were detected in 26 lesions from 20 patients, including 18 carcinomas, 6 dysplasia-associated masses, 1 flat dysplasia, and 1 lymph node metastasis. In two cases, identical p53 mutations were observed in both carcinoma and adjacent dysplasia. Missense mutations causing amino acid substitutions as well as nonsense mutations resulting in premature stop codons were seen. Tandem mutations, in which more than 1 sequence alteration occurred on the same allele of p53, were also detected. Point mutation was accompanied by loss of the other p53 allele in 8 of 10 patients informative for both loss of heterozygosity and mutation assays. CONCLUSIONS These findings suggest that inactivation of p53 by mutation and loss of heterozygosity is a common mechanism of malignant transformation in ulcerative colitis. They also imply that in contrast to sporadic colorectal carcinoma, ulcerative colitis-associated neoplastic progression may involve p53 inactivation at relatively early, noninvasive stages.

Effects of retinoic acid on the growth and morphology of hamster tracheal epithelial cells in primary culture

SummaryHamster tracheal epithelial cells were grown in primary culture on a collagen gel substrate in hormone-supplemented serum-free Ham’s F12 medium with 10-8 M retinoic acid (RA +), or without retinoic acid (RA -). On days 1 and 2, the colonies were composed of large (secretory) cells and lesser numbers of small (basal) cells; ciliated cells were rare. At these times, cell number, thymidine incorporation, and total labelling indices (small and large cells, combined) were similar in RA+ and RA-cultures, but the large cells became flat in RA-medium on day 2. On days 3–5, thymidine incorporation and total labelling indices were less in RA-than RA+ cultures, and on days 4–6, cell numbers were decreased in RA-cultures. On day 3, the large cells of the RA-colonies had flattened further and clusters of small basal cells had formed. On day 4, the RA+ colonies were composed of densely-packed cuboidal secretory cells, small basal cells were inconspicuous; the total labelling index was about 27%. The RA-colonies were composed of large flat secretory cells and numerous small basal cells which were clustered in groups; the total labelling index was about 7%. Since large and small cells could be discriminated by size in RA-colonies, a labelling index was generated based on cell size. On days 2, 3 and 4, the labelling index of the small basal cells in the RA-colonies was 44%, 43% and 24% respectively, whereas the labelling index of the large secretory cells fell rapidly over the same period (56%, 14% and 2%). On days 5 and 6, the cuboidal secretory cells in the RA + cultures had differentiated further and the cells were stratified focally. Some new ciliated cells had formed on day 6. In RA cultures, mucous granules were not observed in the large flat cells and ciliated cells were not seen. The total labelling indices were 11% and 0.35% in RA+ cultures, and 0.5% and 0.25% in RA-cultures on days 5 and 6, respectively.The study shows that the target cell for vitamin A in the hamster tracheal epithelium is the secretory (mucous) cell. When retinoic acid was deficient, the secretory cells flattened and their capacity to divide was greatly diminished. Since the basal cells continued to replicate when the secretory cells did not, the population density of the basal cells increased disproportionally, which could be interpreted erroneously as a “basal cell hyperplasia.” Real hyperplasia and epidermoid metaplasia were late secondary events which occurred in this study following focal disintegration of the epithelial cell sheet. Then the large secretory cells became keratinized and expressed an epidermoid phenotype.

Changes in Secretory Cells of Hamster Tracheal Epithelium in Response to Acute Sublethal Injury: A Quantitative Study

Secretory cells are reported to rapidly increase in number when the tracheobronchial epithelium is irritated. We suggest that this acute change results from accumulation of secretion granules within specialized albeit unobstrusive secretory cells rather than from conversion of undifferentiated cells to secretory cells of the production of new cells by mitosis. This hypothesis was tested in hamster tracheal epithelium 6, 12, and 24 hr after intratracheal instillation of elastase in saline or saline alone. Basal, secretory, and ciliated cells and cells of indeterminate character were quantified by counting 1400 cells per hamster in 2 micrometers thick glycol methacrylate sections stained with periodic acid-Schiff (PAS)-lead hematoxylin. Secretory cells were characterized and quantified depending upon distribution patterns of the PAS-positive secretion granules and on the amount of intracellular secretion product. Thirty-two hamsters were studied; half of them received colchicine 6 hr prior to tissue sampling. Over 24 hr the percentage of ciliated cells showed no significant change and no morphological evidence of alteration was observed in this cell population; thus ciliated cells were excluded from the analysis. When the ciliated cell population was excluded, control and experimental values for the different cell categories remained statistically constant, if unavoidable classification errors at the light microscopic level were compensated for on the basis of ultrastructural features. Average control values for the non-ciliated cell population were: mitotic index 0.030, basal cells 27.8%, secretory cells 57.9%, and cells of indeterminate character 14.3%. Secretory product was decreased in secretory cells at 6 hr but thereafter secretion accumulated in these cells. At 24 hr the overall number of secretory cells appeared to be increased. However, the increase was apparent and not real and was accounted for by accumulation of secretion granules in preexisting but previously unobtrusive secretory cells.

Modulation of glycogen stores in epithelial cells during airway development in Syrian golden hamsters: a histochemical study comparing concanavalin A binding with the periodic acid-Schiff reaction.

We studied glycogen storage in the developing airway epithelium of Syrian golden hamsters from gestational Day 11 to neonatal Day 2 using concanavalin A (ConA) staining as an adjunct approach to the periodic acid-Schiff (PAS) reaction. One hundred and fourteen fetuses and neonates were fixed in 4% formaldehyde-1% glutaraldehyde, 6% mercuric chloride-1% sodium acetate-0.1% glutaraldehyde, and 95% ethanol, embedded in paraffin, and stained with ConA-horseradish peroxidase conjugate as well as with PAS. ConA staining was abolished by alpha-glucosidase digestion or by pre-treatment with periodic acid, demonstrating that ConA bound to glycogen. In tissues fixed with mercury and/or aldehydes, ConA staining was greatly enhanced by pepsin digestion. Airway glycogen stores, revealed by ConA and PAS, fluctuated during development. At first all the undifferentiated epithelial cells contained abundant glycogen. Then, coincident with the appearance of the first endocrine cells, the glycogen stores were depleted. Thereafter, glycogen accumulated in pre-secretory and basal cells until birth, but by 2 days after birth the glycogen stores were again depleted. The initial depletion of glycogen followed by repletion was observed at all levels of the conducting airways; changes in the trachea preceded those in the bronchi and bronchioles by 1 and 2 days, respectively.

Histogenesis and morphogenesis of epidermoid metaplasia in hamster tracheal organ explant culture

SummaryThe formation of epidermoid metaplasia was studied in hamster tracheal epithelium in long-term serum-free organ expiant culture. Explants were cultured up to 5 weeks in CMRL 1066 with antibiotics and amphotericin B. At 3 weeks there were rare small foci of epidermoid metaplasia and they became larger and more numerous at 4 and 5 weeks. Three dimensional reconstructions from serial sections demonstrated that the small deep-seated foci were discrete and did not reach the epithelial surface, whereas the larger foci were expansive and involved the full thickness of the expiant epithelium. Each small focus consisted of a few swollen electron-lucent basal cells attached to the basal lamina, covered by a layer of flattened electron-dense secretory cells which formed a tightfitting cap over the basal cells. The altered secretory cells displayed moderately well-developed rough endoplasmic reticulum and tonofilament bundles. During the early stages of formation the deepseated metaplastic foci were completely covered by a layer of normal appearing cuboidal to low-columnar secretory and ciliated cells. Expansion of the metaplastic foci occurred by addition of flattened , electron-dense secretory cells to the cap so that multiple layers of altered secretory cells covered a core of basal cells, analagous to the structure of an onion. The secretory cells became cornified and with time the foci broke through the columnar mucociliary surface layer. In well-advanced foci, the uppermost cornified squames (metaplastic secretory cells) exfoliated into the trachéal lumen. The study emphasizes similarities and differences between the morphogenesis and histogenesis of epidermoid metaplasia in vivo and in vitro. In vivo, epidermoid metaplasia presents as a full-thickness lesion from its onset, whereas in vitro, the lesions first arise deep in the epithelium and only become full-thickness after an extended period of time. This difference may be explained by the fact that in vitro, the concentration gradients of nutrients and other substances across the thickness of the epithelium are the reverse of such gradients in vivo. However the histogenesis of the lesions is similar in vivo and in vitro and in both cases secretory cells make up a large part of the epidermoid metaplastic foci. The altered secretory cells express an epidermoid phenotype while retaining some characteristics of secretory specialization.

Explant organ culture of hamster alimentary tract epithelium.

Adult Syrian Golden hamster alimentary tract maintained as explants in organ culture was studied using the model system for hamster pancreas described by Resau et al. (1983a). Explants of esophagus, stomach, duodenum and colon were maintained in organ culture on Gelfoam® sponge rafts in a high-oxygen atmosphere with serum-supplemented CMRL-1066 medium. All of the tissues were observed to show evidence of sublethal acute cell injury during the first several days of culture. Subsequently, the epithelial tissues recovered from this injury, repopulated the denuded areas of the explants and replicated within the sponge matrix. Explants were maintained in a differentiated state for 30+ days and sampled for morphology to examine the process of cell injury, repair, differentiation and replication which occurs in mucosal epithelia. The percentage of basement membrane covered by epithelia in the explants from various tissues was compared to the level of LDH in the media to reveal the relationship between viability determined by biochemical and by morphological methods. Restitution of the mucosal surface occurred in all of the explants. We conclude that adequate populations of replicating cells are maintained within the epithelium of the hamster alimentary tract tissues in vitro so that restitution can occur through migration and subsequent differentiation of the epithelial cells within the mucosa of the explants.