A candidate precursor to serous carcinoma that originates in the distal fallopian tube.

The tubal fimbria is a common site of origin for early (tubal intraepithelial carcinoma or TIC) serous carcinomas in women with familial BRCA1 or 2 mutations (BRCA+). Somatic p53 tumour suppressor gene mutations in these tumours suggest a pathogenesis involving DNA damage, p53 mutation, and progressive loss of cell cycle control. We recently identified foci of strong p53 immunostaining-termed 'p53 signatures'-in benign tubal mucosa from BRCA+ women. To examine the relationship between p53 signatures and TIC, we compared location (fimbria vs ampulla), cell type (ciliated vs secretory), evidence of DNA damage, and p53 mutation status between the two entities. p53 signatures were equally common in non-neoplastic tubes from BRCA+ women and controls, but more frequently present (53%) and multifocal (67%) in fallopian tubes also containing TIC. Like prior studies of TIC, p53 signatures predominated in the fimbriae (80-100%) and targeted secretory cells (HMFG2 + /p73-), with evidence of DNA damage by co-localization of gamma-H2AX. Laser-capture microdissected and polymerase chain reaction-amplified DNA revealed reproducible p53 mutations in eight of 14 fully-analysed p53 signatures and all of the 12 TICs; TICs and their associated ovarian carcinomas shared identical mutations. In one case, a contiguous p53 signature and TIC shared the same mutation. Morphological intermediates between the two, with p53 mutations and moderate proliferative activity, were also seen. This is the first report of an early and distinct alteration in non-neoplastic upper genital tract mucosa that fulfils many requirements for a precursor to pelvic serous cancer. The p53 signature and its malignant counterpart (TIC) underline the significance of the fimbria, both as a candidate site for serous carcinogenesis and as a target for future research on the early detection and prevention of this disease.

Histologic and immunophenotypic classification of cervical carcinomas by expression of the p53 homologue p63: a study of 250 cases.

Recent studies of the p53 homologue p63 indicate that this gene is preferentially expressed in basal and immature cervical squamous epithelium. This study correlated p63 expression with morphologic phenotype and human papillomavirus (HPV) type in a wide range of cervical neoplasms. Two hundred fifty cases of cervical carcinoma, including squamous cell carcinoma (SCCA; n = 178), adenocarcinoma (ADCA; n = 28), adenosquamous carcinoma (ASCA; n = 8), neuroendocrine carcinoma (NECA; n = 15), and other variant or mixed types (n = 21) were studied. Ninety-seven percent of SCCA, 0% of ADCA, and 0% of SCUC showed strong (>75% v <30%) positivity for p63 (P<.001). p63 sharply distinguished SCCA (p63+) from ADCA (p63-), Large-cell, poorly differentiated carcinomas were distinguished as putative glandular (glassy cell) or squamous (lymphoepithelial-like or spindle cell) types based on p63 staining. Eight (73%) of 11 neuroendocrine tumors tested were chromogranin positive; all showed no or low (<30%) levels of p63 immunostaining. Absence of p63 was also associated with a subset of nonneuroendocrine undifferentiated carcinomas. Transitions from squamous to columnar or undifferentiated morphology coincided with loss of p63 expression. A strong association between HPV 16 and p63 positivity was identified because of the colocalization of both within tumors of squamous phenotype. p63 is a powerful marker for squamous differentiation and, when diffusely expressed, excludes a glandular or neuroendocrine differentiation. p63 may be useful for differentiating pure squamous or glandular from adenosquamous carcinomas, tracking shifts in differentiation within tumors, supporting (by its absence) the diagnosis of neuroendocrine carcinomas, and clarifying the spectrum of poorly differentiated carcinomas lacking either squamous or neuroendocrine differentiation.

Expression of the p53 homologue p63 in early cervical neoplasia.

BACKGROUND: p63, a homologue of the tumor suppressor gene p53, is expressed in embryonic, adult murine, and human basal squamous epithelium and encodes both transactivating and dominant negative transcript isoforms. Mouse embryos functionally deficient in p63 fail to replenish basal squamous epithelial cells, resulting in multiple defects that include absent genital squamous epithelium. This study investigated the expression of p63 in the human cervical transformation zone and early cervical neoplasia. METHODS: Tissue localization of p63 was determined by immunohistochemistry in a wide range of epithelia. A correlation was also made between p63 expression and squamous basal cell (keratin 14), endocervical columnar cell (mucicarmine), and cell-cycle specific (Ki-67) markers. RESULTS: p63 expression by immunostaining delineated basal and parabasal cells of maturing ectocervical squamous mucosa, squamous metaplasia in the cervix, and basal and subcolumnar cells of the cervical transformation zone. In atrophic epithelia immunostaining for p63 was present in all cell strata. In early cervical neoplasia, p63 expression was inversely correlated with both squamous cell maturation and nonsquamous differentiation in CIN. This biomarker also identified basal cells in a subset of preinvasive cervical neoplasms with endocervical cell differentiation that were bcl-2 and keratin 14 negative. CONCLUSIONS: In the lower female genital tract, p63 is preferentially expressed in immature cells of squamous lineage and is not linked to cell proliferation. The broader range of p63 expression relevant to keratin 14 and bcl-2 indicates that p63 may identify additional subsets of benign and neoplastic epithelial basal cells in the cervical transformation zone and may be useful in studying cell differentiation in the early stages of neoplastic change in this region.

Identification of a basal/reserve cell immunophenotype in benign and neoplastic endometrium: a study with the p53 homologue p63.

BACKGROUND: Metaplastic differentiation, including squamous, mucinous, and tubal (ciliated), is common in both benign and neoplastic endometrium, and the cell of origin for this pathway is poorly understood. In this study, expression of a marker for basal and reserve cells in cervical squamous mucosa, designated p63, was investigated in a spectrum of endometrial alterations. METHODS: One hundred ninety different endometria from 132 patients were examined, including fetal (6), premenarchal (3), benign cyclic (29) and noncyclic (54), hyperplastic (14), and neoplastic (93) endometrial glandular epithelia. The latter included conventional endometrioid carcinomas with and without mucinous, ciliated, and squamous metaplasia, and uterine papillary serous carcinoma (UPSC). RESULTS: p63 expression was identified in basal/subcolumnar cells in the fetal endometrium in a distribution similar to that in basal/reserve cells of the cervix. Staining was confined to individual scattered basal and suprabasal cells in cycling endometrium. In polyps and postmenopausal endometria, focal clusters of p63-positive cells were identified in inactive glands or surface epithelium. Metaplastic (squamous or mucinous) epithelia, either alone or in conjunction with hyperplasias or carcinomas, exhibited the most intense staining, primarily in basal or subcolumnar cells. In some cases, immediately adjacent nonmetaplastic columnar epithelium also stained positive. UPSCs contained only rare scattered p63-positive cells. CONCLUSIONS: Cells with a basal or reserve cell phenotype exist in the endometrium during fetal life, are not conspicuous during the reproductive years, but may emerge during shifts in differentiation. Whether these cells signify specialized multipotential endometrial cells is not clear, but the similarity of these cells to basal/reserve cells of the cervix and their association with neoplasia merit further study.

Adenoid basal carcinomas of the cervix: a unique morphological evolution with cell cycle correlates.

Adenoid basal carcinoma (ABC) is a rare cervical carcinoma of postmenopausal women composed of small basal-type (basaloid) cells with focal endocervical ("adenoid") differentiation. ABCs are associated with high-grade squamous intraepithelial lesions (HSIL) and contain integrated human papillomavirus type 16 DNA. However, ABCs have a favorable prognosis and do not metastasize. Five (5) ABCs were analyzed histologically for a marker distinguishing basal/ squamous from columnar (adenoid) differentiation (p63) and cell cycle activity (Ki-67), and compared with 20 cervical (CC) carcinomas. In contrast to other CCs, ABCs contained 4 distinct components, including (1) a classic HSIL; (2) a limited invasive component with squamoid maturation, often with a discrete layer of peripheral basal cells; (3) outgrowth of small basal cells from either HSIL or squamoid areas; (4) focal endocervical (adenoid) differentiation. ABCs showed distinct differences in cell cycle activity relative to CCs. Ki-67 positivity was high in associated HSILs but remained high and concentrated in the suprabasal cells of the invasive squamoid component of ABC. Moreover, proliferative index was variable to sharply reduced in areas of basaloid and adenoid differentiation, in contrast to conventional CCs. ABC is a unique neoplasm, not only by its transition through multiple phenotypes during invasion, but also by a proliferative index that is high in more mature neoplastic cells during the infiltrative process and reduced with progressive basal differentiation. The precise mechanism underlying this unique process of tumor evolution is unclear. However, the postmenopausal status of these patients suggests that host factors related to aging may influence tumor evolution and morphology after HPV 16 infection.

Cyclophilin B trafficking through the secretory pathway is altered by binding of cyclosporin A.

Cyclophilin B is targeted to the secretory pathway via an endoplasmic reticulum signal sequence. We analyzed the localization and trafficking of endogenous and transfected cyclophilin B in mammalian cells. Cyclophilin B accumulates both in the endoplasmic reticulum and in complexes on the plasma membrane. The immunosuppressant cyclosporin A specifically mobilizes cyclophilin B from the endoplasmic reticulum, and promotes the secretion of cyclophilin B into the medium. We suggest that cyclosporin A competes with endogenous plasma membrane proteins for association with cyclophilin B in the secretory pathway. These findings argue in favor of a role for cyclophilin B as a chaperone to proteins destined for the plasma membrane, rather than solely as a proline isomerase functioning within the endoplasmic reticulum.

Cyclosporin A, the cyclophilin class of peptidylprolyl isomerases, and blockade of T cell signal transduction.

Cyclosporin A, the major immunosuppressive drug in transplantation, and the more potent therapeutic drug candidate, FK506, have led to the discovery of two superfamilies of immunosuppressant binding proteins, the cyclophilins and the FK binding proteins. These proteins, enzymes with high kcat values for isomerization of X-Pro bonds in peptides and protein substrates, are distributed in all cell compartments where protein folding normally occurs. It is likely that they play major roles in the protein folding and protein trafficking in the cell. It is also likely that they have been suborned in T cells by the immunosuppressant drugs that are potent pseudosubstrate ligands that selectively block the signal transduction cascade. The discovery of the inhibition of protein phosphatase 2B (calcineurin) by the drug-immunophilin complex (CsA-CyP or FK506-FKBP) provides evidence for a specific downstream target of the drug-immunophilin complexes and may prompt a search for endogenous ligands of cyclophilin and FKBP that may effect signal transduction regulation. The molecular insights gained over a short time in this area have been remarkable; they promise to elucidate the steps in T cell activation and delineate new targets for immunosuppressive therapy.

Human cyclophilin B: a second cyclophilin gene encodes a peptidyl-prolyl isomerase with a signal sequence.

We report the cloning and characterization of a cDNA encoding a second human cyclosporin A-binding protein (hCyPB). Homology analyses reveal that hCyPB is a member of the cyclophilin B (CyPB) family, which includes yeast CyPB, Drosophila nina A, and rat cyclophilin-like protein. This family is distinguished from the cyclophilin A (CyPA) family by the presence of endoplasmic reticulum (ER)-directed signal sequences. hCyPB has a hydrophobic leader sequence not found in hCyPA, and its first 25 amino acids are removed upon expression in Escherichia coli. Moreover, we show that hCyPB is a peptidyl-prolyl cis-trans isomerase which can be inhibited by cyclosporin A. These observations suggest that other members of the CyPB family will have similar enzymatic properties. Sequence comparisons of the CyPB proteins show a central, 165-amino acid peptidyl-prolyl isomerase and cyclosporin A-binding domain, flanked by variable N-terminal and C-terminal domains. These two variable regions may impart compartmental specificity and regulation to this family of cyclophilin proteins containing the conserved core domain. Northern blot analyses show that hCyPB mRNA is expressed in the Jurkat T-cell line, consistent with its possible target role in cyclosporin A-mediated immunosuppression.

Nuclear lamin LI of Xenopus laevis: cDNA cloning, amino acid sequence and binding specificity of a member of the lamin B subfamily.

Lamins are karyoskeletal proteins associated with the nuclear envelope which can be divided into two groups, i.e. the type A lamins of near neutral pI and the more acidic lamins, including mammalian lamin B. We have isolated cDNA clones encoding a representative of the type B subfamily from Xenopus laevis, and have deduced its amino acid sequence from the coding portion of the approximately 2.9 kb mRNA. The polypeptide (mol. wt 66,433) is identified as a typical lamin by its homology to Xenopus human type A lamins, but detailed sequence comparison shows that LI is less related to Xenopus lamin A than the latter is to human lamin A. The conformation predicted for LI conforms to the general model of lamins and intermediate filament proteins and is characterized by an extended central alpha-helical coiled coil domain, flanked by non-alpha-helical domains, i.e. a relatively short N-terminal head and a long C-terminal tail. As in lamins A and C, the head of lamin LI is positively charged and the tail presents a similar C-terminal pentapeptide, a putative nuclear accumulation signal, a very negatively charged region and a number of short regions that are highly homologous in all lamins. However, LI differs from the type A lamins by the absence of the oligo-histidine stretch and a di-proline motif in the tail region and by a significantly lower number of identical amino acid positions.(ABSTRACT TRUNCATED AT 250 WORDS)

Homologies in both primary and secondary structure between nuclear envelope and intermediate filament proteins.

The A, B and C lamins are the major proteins of the nuclear envelope. The complete nucleotide sequence of the coding region of the A and C lamins shows that these proteins are identical except for their carboxy termini. The most prominent structural feature of both lamins is an alpha-helical region of repeating heptads of amino acids that shows striking homology with the entire family of cytoplasmic intermediate filament proteins. These features suggest that the nuclear envelope is made up of a network of coiled-coil polymers.

The redistribution of a conserved nuclear envelope protein during the cell cycle suggests a pathway for chromosome condensation.

We describe a human autoantiserum that recognizes specific determinants present both on the nuclear envelope of interphase cells and the periphery of metaphase chromosomes. These determinants are highly conserved through evolution and present on a protein with an apparent molecular weight of 33,000. This 33 kd protein, which we call "perichromin," appears to be directly or indirectly bound to both interphase and metaphase DNA. Studies of the transformation of perichromin from a nuclear envelope association to a perichromosomal position during prophase suggests a pathway for chromosome organization throughout the cell cycle.

Autoimmune response directed against conserved determinants of nuclear envelope proteins in a patient with linear scleroderma.

We have studied the autoantibodies in the serum of a patient with linear scleroderma that specifically recognize the nuclear envelope of cultured cells. These antibodies bind to conserved determinants of nuclear lamins, the predominant mammalian nuclear envelope proteins. Of the three mammalian nuclear lamin proteins (970, P68, and P60), only P70 and P60 bind the autoantibodies. In addition, two proteins of the Drosophila embryonic nuclear matrix, P70 and P68, bind these autoantibodies. We have used nuclear matrices to isolate the autoantibodies from the patient's serum that react to the nuclear lamins. At least three different IgG heavy chains were found to be involved in this autoimmune response to nuclear lamins, indicating that this response is not due to the expansion of a single B-cell clone.