Epstein-Barr virus and gastric carcinoma

Immunohistopathological staining for p53, PCNA and Ki67 was performed in 120 specimens from previously untreated laryngeal carcinomas using the avidin-biotin method with peroxidase as a marker enzyme and diaminobenzidine as a chromogen. A 5-grade staining score system was used. Statistically significant correlations (Chi-square) were seen between T- and N-stage and histopathological grading. p53 and Ki67 scoring correlated with T- and N-stage whereas PCNA with T-stage. All staining correlated with histopathological grading. The score of staining for p53, PCNA and Ki67 correlated with each other. The patients with recurrences within 3 years had mainly carcinomas with higher staining scores. Using Chi-square analysis the p53, PCNA and Ki67 staining scores were also independent prognostic indicators.     

Localization and characterization of a chromosome 11 tumor suppressor gene using organotypic raft cultures

The development and progression of human cancer often involves the inactivation of tumor suppressor gene function. Alterations in human chromosome 11 during the development of human cutaneous squamous cell carcinoma suggest the presence of a tumor suppressor gene on this chromosome. Moreover, previous studies in our laboratory demonstrated the presence of a functional tumor suppressor gene on chromosome 11 for the human cutaneous squamous cell carcinoma cell line A388.6TG.c2. In this investigation, we have used organotypic culturing of epithelial cells as a novel in vitro assay for tumor suppression. A388.6TG.c2 and control cells form an abnormal stratified epithelium of 8-12 layers when cultivated on organotypic rafts. In contrast, the chromosome 11 microcell hybrids, HMC 100p4B and HMC 100p5A, form an epithelium of only two to three cell layers. This in vitro growth suppression of the chromosome 11 microcell hybrids in the organotypic rafts correlates well with our previous in vivo skin graft experiments. Comparison of the proliferation and apoptotic indices of cell lines grown on the organotypic rafts suggests that the tumor suppressor gene on chromosome 11 has restricted the ability of the microcell hybrids to stratify but has not significantly altered their ability to undergo cell division or programmed cell death. Furthermore, flow cytometric analysis of cells grown on organotypic raft cultures suggests that the chromosome 11 microcell hybrids are actively progressing through the cell cycle rather than arrested in a particular stage. We have used this novel application of organotypic raft cultures to further localize the chromosome 11 tumor suppressor gene. Introduction of a single der(11)t(X;11) chromosome lacking most of the long arm of chromosome 11 into A388.6TG.c2 does not affect growth on organotypic raft cultures. These data suggest the tumor suppressor gene maps to the long arm of chromosome 11 in the region of 11q13-qter.     

Evaluation of topical gene therapy for head and neck squamous cell carcinoma in an organotypic model

The organotypic (raft) culture system has been shown to be a useful model for examining the effects of biochemical manipulations on various epithelial cell types, using in vitro conditions that simulate the in vivo environment of the tissue of origin. To investigate this method as a model for topical gene therapy, we cultured the oral head and neck squamous cell carcinoma cell line TR146 on fibroblast-containing collagen gels at the air-medium interface and assessed the efficiency of transduction of a topically applied adenoviral vector containing beta-galactosidase cDNA. Diffuse expression of -galactosidase activity in multiple cell layers demonstrated effective penetration of the vector. Transduction efficiency and therapeutic activity of a replication-defective recombinant adenovirus containing wild-type p53 cDNA linked to a FLAG marker (AdCMV-p53-FLAG) were then assessed in TR146 organotypic cultures transduced by topical application. Twenty-four, 48, and 72 h after transduction, the cultures were harvested, and residual cell number and FLAG peptide expression were determined. The number of cells in p53 transduced cultures was significantly reduced in comparison to controls at all three time points (P < 0.001), which resulted from the induction of apoptosis as determined by in situ DNA end labeling. In addition, the FLAG peptide was expressed diffusely in the residual cells, further confirming effective transduction and expression of the exogenous gene products throughout multiple layers. We conclude that the organotypic culture is an effective in vitro model for assessing the efficacy of topically applied gene therapy on head and neck squamous carcinomas and premalignancies.     

Apocrine adenosis: a precursor of aggressive breast cancer?

AIM: To investigate overexpression of c-erbB2, expression of the p53 protein product and proliferation rates in benign breast lesions with specific reference to apocrine adenosis. METHODS: Twenty one cases of apocrine adenosis were stained with monoclonal antibodies to p185, the protein product of the c-erbB2 oncogene, the protein product of the p53 tumour suppressor gene and to the cell cycle related protein Ki67. Three cases were associated with concomitant ductal carcinoma in situ of large cell type and two were associated with invasive tubular or cribriform carcinoma. RESULTS: Twelve (57.1%) cases showed membrane staining for c-erbB2 oncoprotein of apocrine cells within sclerosing adenosis and six (28.6%) had occasional p53 protein positive cells. One case not associated with carcinoma showed extensive staining of apocrine metaplasia outside the area of apocrine adenosis. The proliferation rate, as measured by Ki67 staining, was increased in some of the lesions and all lesions showed at least some of the cells to be in the cell cycle. CONCLUSIONS: The expression of abnormal oncogene products and increased proliferation in some of these apocrine lesions questions the supposed degenerative nature of the atypia seen in such cases and suggests that there may be an association between these lesions and large cell ductal carcinoma in situ and hence invasive carcinoma.     

Anticonvulsant drug effects on spontaneous thalamocortical rhythms in vitro: ethosuximide, trimethadione, and dimethadione

The state of the basement membrane (BM) was investigated in the normal epithelium, dysplasia/carcinoma in situ (CIS) and invasive carcinoma of the uterine cervix as well as metastatic lymph nodes by performing immunohistological staining for the presence of laminin (LN) and type IV collagen (CIV) and periodic acid-methenamine silver (PAM) staining of the BM. Moreover, to clarify the relationship between the epithelial cells and the BM, simultaneous staining for proliferating cell nuclear antigen (PCNA) was performed on the repaired tissue after punch biopsy. In the normal tissues, positive linear staining was observed beneath the epithelium with both PAM staining and LN.C IV staining. Some of the cases of dysplasia/CIS were continuously positive with PAM staining. However, there were sites which showed weakening and disruption of the continuity of positive staining for LN and C IV. As a function of the histological type of invasive carcinoma, the intensity of staining for LN and C IV decreased in the order of keratinizing (K), large cell non-keratinizing (LNK) and small cell non-keratinizing (SNK) histological types. However, no correlation was found between the stage of the carcinoma and the intensity of staining. In addition, the staining of the metastatic lymph nodes was similar to that of the primary lesion, and cancer foci that were in direct contact with lymphocytes were also stained for LN and C IV. During the process of reformation of the BM, LN and C IV were already present at the time when the epithelial interstitium was still negative for PAM staining. Moreover, at the time when the epithelial basal cells were PCNA-positive, LN and C IV could not be detected, and, conversely, when PCNA was negative, LN and C IV were detected. The BM is a structure which is formed when the interstitium is in a static state, and it was surmised that the production of LN and C IV is carried out by epithelial cells, including cancerous cells. Furthermore, it was surmised that the BM is not a structure which can prevent cancer invasion and the discontinuity or intermittency of staining for LN and C IV in cancers is a result of changes in the capacity to produce the BM components due to canceration of the cells.     

Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53

The p53 tumor suppressor gene has been shown to play an important role in determining cell fate. Overexpression of wild-type p53 in tumor cells has been shown to lead to growth arrest or apoptosis. Previous studies in fibroblasts have provided indirect evidence for a link between p53 and senescence. Here we show, using an inducible p53 expression system, that wild-type p53 overexpression in EJ bladder carcinoma cells, which have lost functional p53, triggers the rapid onset of G1 and G2/M growth arrest associated with p21 up-regulation and repression of mitotic cyclins (cyclin A and B) and cdc2. Growth arrest in response to p53 induction became irreversible within 48-72 h, with cells exhibiting morphological features as well as specific biochemical and ultrastructural markers of the senescent phenotype. These findings provide direct evidence that p53 overexpression can activate the rapid onset of senescence in tumor cells.     

Spontaneous in vitro immortalization of breast epithelial cells from a patient with Li-Fraumeni syndrome

Individuals with germ line mutations in the p53 gene, such as Li-Fraumeni syndrome (LFS), have an increased occurrence of many types of cancer, including an unusually high incidence of breast cancer. This report documents that normal breast epithelial cells obtained from a patient with LFS (with a mutation at codon 133 of the p53 gene) spontaneously immortalized in cell culture while the breast stromal fibroblasts from this same patient did not. Spontaneous immortalization of human cells in vitro is an extremely rare event. This is the first documented case of the spontaneous immortalization of breast epithelial cells from a patient with LFS in culture. LFS patient breast stromal fibroblasts infected with a retroviral vector containing human papillomavirus type 16 E7 alone were able to immortalize, whereas stromal cells obtained from patients with wild-type p53, similarly infected with human papillomavirus type 16 E7, did not. The present results indicate a protective role of normal pRb-like functions in breast stromal fibroblasts but not in breast epithelial cells and reinforces an important role of wild-type p53 in the regulation of the normal growth and development of breast epithelial tissue.     

Low frequency epithelial cells in bone marrow aspirates from prostate carcinoma patients are cytogenetically aberrant

BACKGROUND: Low frequency epithelial cells occur in bone marrow aspirates of 25-50% of patients with locally confined prostate carcinoma. It is assumed that bone marrow epithelial cells derive from the primary tumor; however, it has not been established unequivocally that they are tumor cells. Immunofluorescence approaches were used to quantify the frequency of epithelial cells in bone marrow aspirates from prostate carcinoma patients and genotypic analyses were used to determine whether they contained numeric aberrations of chromosomes 1, 7, and 8. METHODS: Epithelial cells in bone marrow aspirates collected after radical prostatectomy were visualized using fluorescence microscopy and fluorophore-linked antibodies against cytokeratin 8,18 (CK) and prostate specific antigen (PSA). Antibodies specific for proliferating nuclear cell antigen (PCNA) were used to evaluate the cycling status of discriminated cells. Copies of chromosomes 1, 7, and 8 in the discriminated epithelial cells were quantified using fluorescence in situ hybridization. RESULTS: CK+ cells were present in bone marrow aspirates from 30 of 66 patients (approximately 45%) at a median frequency of 1.4 CK+ cells/10(5) mononuclear cells. Few CK+ epithelial cells in the bone marrow aspirates coexpressed PSA and none of the CK+ cells expressed PCNA. Approximately 70-75% of the CK+ cells contained 7 and 8 aneusomies. Gains of chromosome 1 occurred in 42% of the CK+ cells. CONCLUSIONS: The majority of CK+ cells in bone marrow aspirates collected after surgery are cytogenetically aberrant, which is consistent with a primary tumor origin. The prevalence and frequency of CK+ cells is independent of tumor stage/grade and androgen treatment.     

One single erythemagenic UV irradiation is more effective in increasing the proliferative activity of melanocytes in melanocytic naevi compared with fractionally applied high doses

The effect of a single irradiation with UV light on the expression of Ki67 antigen, topoisomerase II alpha, proliferating cell nuclear antigen (PCNA), the melanocyte activation marker HMB-45 and protein p53 in melanocytic naevi was investigated 1 week after application of a single erythemagenic UV dose and after daily exposures with suberythemagenic doses over 4-6 weeks. To assess the effect of UV irradiation, one half of each naevus was shielded with black tape during the UV exposure, and the irradiated part and the non-irradiated parts were evaluated separately. Except for HMB-45, a double staining procedure was performed to distinguish between labelled melanocytes and keratinocytes. After semiquantitative assessment of the staining signal the irradiated part was compared with the non-irradiated part of the same naevus. Morphological changes and an enhanced proliferative/ reparative activity in melanocytes were much more frequent in the naevi irradiated with a single erythemagenic UV dose than in those given repeated suberythemagenic doses. In addition, the keratinocytes showed an increased labelling for PCNA and p53 after the single irradiation. These data may support the importance of intermittent UV exposure and sunburns in the development of both benign and malignant melanocytic lesions.     

Effects of sodium acetate on rat bone-nodule formation and mineralization in vitro

A new cell line, FR-car, has been established from a biopsy of a low-grade human cervical squamous intraepithelial lesion (SIL). We confirmed the epithelial origin of the cells by keratin staining using polykeratin, AE1/AE3 and CAM 5.2 antibodies. Sixty percent to 80% of the cultured cells stained positive for proliferative cell nuclear antigen (PCNA) and Ki-67. There was no overexpression of p53. Karyotyping revealed that the cell line was hypodiploid with clonal abnormalities on chromosome 6 and 16. Sections of a biopsy adjacent to the lesion from which the culture was initiated tested positive for human papillomavirus (HPV) 18 DNA by the polymerase chain reaction, but cultured cells tested at several passages were HPV-negative by either type-specific or consensus PCRs. This HPV-negative SIL line may be useful in studies into the cell biology of dysplastic epithelium.     

Failure of complex supplementation of minimal cultures to elicit a shift-up response in Pseudomonas putida

Primary cell cultures are established from 8-day quail embryo livers. During the first three days the culture is made up of areas of epithelial-like cells and scattered fibroblasts. The cytoplasm of the epithelial cells shows a high glycogen content as detected by the PAS reaction controlled with salivary amylase digestion. During the following days an important increase in the number of fibroblastic cells is observed. After 6-7 days of cultivation, the epithelial cells have disappeared and the culture is entirely fibroblastic. PAS technique does not show any trace of glycogen in these cultures which have been prolonged up to 45 days. Six-to 45-day primary cultures entirely made up of fibroblasts were associated with hepatic or pulmonary mesenchyme in organotypic culture for 3-4 days. In some cases the explant was first cultivated in vitro for 2 days and then grafted into a 5-day-old chick embryo on the chorioallantoic membrane for 6 days. In the secondary cultures hepatocytes showing an epithelial arrangement and a high glycogen content were observed. It appears from this observation that some of the primary culture fibroblasts are in fact dedifferentiated parenchymal cells. Such a dedifferentiation is a reversible phenomenon since the cells retain the ability to express their initial determination if they are placed in convenient environmental conditions. The role of the specific tissular arrangement in the stability of the differentiated state is discussed.     

Relationship between p21 expression and mutation of the p53 tumor suppressor gene in normal and malignant ovarian epithelial cells

In many cell types, p53-mediated growth inhibition is dependent on induction of p21, which is an inhibitor of cyclin-dependent kinases that are required for cell cycle progression. Failure of mutant p53 proteins to transactivate p21 may lead to uncontrolled proliferation. Because many ovarian cancers have mutations in the p53 gene, we examined p21 levels in normal and malignant ovarian epithelial cells to determine whether p21 expression is dependent on wild-type p53. Normal ovarian epithelial cells and two ovarian cancer cell lines with wild-type p53 expressed readily detectable levels of p21, whereas in p53 null and mutant cell lines, expression of p21 was diminished strikingly. A correlation between the status of the p53 gene and p21 expression also was noted in 23 primary epithelial ovarian cancers. Normal levels of p21 RNA were seen in 4/7 (57%) cancers with wild-type p53, whereas 14/16 (88%) cancers with mutant p53 had reduced p21 expression (P < 0.05). In addition, we found that lambda-irradiation of normal and malignant ovarian epithelial cells with wild-type, but not mutant, p53 resulted in induction of p21. These data are suggestive that induction of p21 is a feature of p53-mediated growth inhibition in normal ovarian epithelial cells. Conversely, mutation of the p53 gene in ovarian cancers usually is associated with decreased p21 expression. The lack of an absolute correlation between p21 expression and the status of the p53 gene in ovarian cancers is consistent with other studies that have suggested that p21 may also be regulated by p53-independent pathways.     

Nuclear accumulation of p21Cip1 at the onset of mitosis: a role at the G2/M-phase transition

Cell cycle arrest in G1 in response to ionizing radiation or senescence is believed to be provoked by inactivation of G1 cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21(Cip1/Waf1/Sdi1). We provide evidence that in addition to exerting negative control of the G1/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G2/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Moreover, synchronized E6-expressing fibroblasts show accelerated entry into mitosis compared to wild-type cells and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Finally, primary embryonic fibroblasts derived from p21-/- mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G2 that may contribute to the implementation of late cell cycle checkpoint controls.     

Comparison of bax, waf1, and IMP dehydrogenase regulation in response to wild-type p53 expression under normal growth conditions

Recently, we demonstrated that downregulation of inosine-5'-monophosphate dehydrogenase (IMPD; IMP:NAD oxidoreductase, EC 1.2.1.14), the rate-limiting enzyme for guanine nucleotide biosynthesis, is required for p53-dependent growth suppression. These studies were performed with cell lines derived from immortal, nontumorigenic fibroblasts that express wild-type p53 conditionally by virtue of a metal-responsive promoter. Here, the p53-dependent properties of the original "p53-inducible" fibroblasts are presented in detail and compared to related properties of epithelial cells that also express wild-type p53 conditionally, but by virtue of a temperature-responsive promoter. Both types of p53-inducible cells were designed to approximate normal physiologic relationships between the host cell and the regulated p53 protein. Together, they were used to investigate expression relationships between IMPD and other p53-responsive genes proposed as mediators of p53-dependent growth suppression. In both types of cells, IMPD activity, protein, and mRNA were consistently coordinately reduced in response to p53 expression. In contrast, mRNAs for waf1, bax, and mdm2 showed disparate patterns of expression, being induced in one conditional cell type, but not the other. This distinction in regulation pattern suggests that under normal growth conditions, unlike IMPD downregulation, bax and waf1 induction is not a rate-determining event for p53-dependent growth suppression.     

Sequential immunotyping and genotyping of tumor cells in bone marrow of cancer patients: a model study

Epithelial cells can be detected in bone marrow or peripheral stem cell preparations of patients with various kinds of cancer and their presence in bone marrow is of prognostic significance. Characterization of these cells has been hampered by their low frequency. Here we present a method that may allow sequential immunophenotyping and genotyping of epithelial cells in bone marrow. To simulate in vivo situations, cells from the colon cancer cell line HT29 were seeded into bone marrow and were first detected by the Fab fragment of the A45-B/B3 anticytokeratin antibody. Expression of Ki67, p53, Her-2/ neu (c-erbB2), and 17-1A could be detected on A45-B/B3-stained cells by immunofluorescence using a fluorescein-labeled anti-mouse immunoglobulin specific for the Fc part of mouse immunoglobulins. Reactivity for all antigens except for Ki67 persisted after A45-B/B3 labeling even when a scoring step for the presence of epithelial cells was performed before proceeding with the immunophenotyping. After immunophenotyping, numerical chromosomal aberrations and amplifications of the Her-2/neu oncogene could be assessed by fluorescence in situ hybridization in the same A45-B/B3-stained cells. This combination of immunophenotyping and genotyping may help in establishing the role of epithelial cells in bone marrow or peripheral stem cell harvests for tumor relapse and formation of metastases.     

Antimicrobial susceptibility of periodontopathic bacteria associated with failing implants

Histological, immunohistochemical, and flow cytometric characteristics of three unusual parotid gland tumors are described. The patients were adult white men with carcinoma ex pleomorphic adenoma, true malignant mixed tumor, and primary parotid gland chondrosarcoma. The carcinoma ex pleomorphic adenoma showed evidence of simultaneous epithelial, myoepithelial, and mesenchymal differentiation by immunohistochemistry. The true malignant mixed tumor exhibited variable positivity for two keratins, vimentin, proliferating cell nuclear antigen, Ki67, and p53. The chondrosarcoma initially stained for vimentin, S100, muscle-specific actin, proliferating cell nuclear antigen, and Ki67, but it lost actin expression in its first recurrence, accompanied by more extensive Ki67 staining. DNA ploidy varied from diploid to aneuploid with intratumoral variation in the carcinosarcoma. S-phase fractions ranged from 2.43% to 13.9%. The findings underscore the diversity of tumors that may be pathogenetically related to, and at times derived from, pleomorphic adenoma.     

p53 and mdm2 are expressed independently during cellular proliferation

We analysed p53 expression during proliferation of serum stimulated Swiss mouse 3T3 cells and of concanavalin A stimulated mouse spleen lymphocytes and correlated it to rate of DNA synthesis and to expression of PCNA. We also analysed mdm2 gene expression, as rising p53 levels during proliferation might require MDM2 protein expression to functionally antagonize p53 mediated growth inhibition. p53 protein synthesis closely paralleled DNA synthesis and PCNA expression, suggesting a direct involvement of p53 in cellular DNA synthesis. mdm2 expression in 3T3 cells could not be correlated with p53 expression and DNA synthesis and was not detected at all in stimulated lymphocytes. We conclude that p53 and mdm2 expression during proliferation are not functionally related and that mdm2 expression is not required for proliferation.