Generation and characterization of human smooth muscle cell lines derived from atherosclerotic plaque.

The study of atherogenesis in humans has been restricted by the limited availability and brief in vitro life span of plaque smooth muscle cells (SMCs). We describe plaque SMC lines with extended life spans generated by the expression of the human papillomavirus (HPV)-16 E6 and E7 genes, which has been shown to extend the life span of normal adult human aortic SMCs. Resulting cell lines (pdSMC1A and 2) demonstrated at least 10-fold increases in life span; pdSMC1A became immortal. The SMC identity of both pdSMC lines was confirmed by SM22 mRNA expression. pdSMC2 were generally diploid but with various structural and numerical alterations; pdSMC1A demonstrated several chromosomal abnormalities, most commonly -Y, +7, -13, anomalies previously reported in both primary pdSMCs and atherosclerotic tissue. Confluent pdSMC2 appeared grossly similar to HPV-16 E6/E7-expressing normal adult aortic SMCs (AASMCs), exhibiting typical SMC morphology/growth patterns; pdSMC1A displayed irregular cell shape/organization with numerous mitotic figures. Dedifferentiation to a synthetic/proliferative phenotype has been hypothesized as a critical step in atherogenesis, because rat neonatal SMCs and adult intimal SMCs exhibit similar gene expression patterns. To confirm that our pdSMC lines likewise express this apparent plaque phenotype, osteopontin, platelet-derived growth factor B, and elastin mRNA levels were determined in pdSMC1A, pdSMC2, and AASMCs. However, no significant increases in osteopontin or platelet-derived growth factor B expression levels were observed in either pdSMC compared with AASMCs. pdSMC2 alone expressed high levels of elastin mRNA. Lower levels of SM22 mRNA in pdSMC1A suggested greater dedifferentiation and/or additional population doublings in pdSMC1A relative to pdSMC2. Both pdSMC lines (particularly 1A) demonstrated high message levels for matrix Gla protein, previously reported to be highly expressed by human neointimal SMCs in vitro. These results describe 2 novel plaque cell lines exhibiting various features of plaque SMC biology; pdSMC2 may represent an earlier plaque SMC phenotype, whereas pdSMC1A may be representative of cells comprising an advanced atherosclerotic lesion.

Endocervical glandular atypia: does a preneoplastic lesion of adenocarcinoma in situ exist?

Some believe endocervical glandular atypia (EGA), purportedly composed of cells that are less atypical than cells of adenocarcinoma in situ (AIS), is a preneoplastic precursor of AIS. We examined 246 neoplastic and nonneoplastic cervical cone biopsy and hysterectomy specimens from 221 patients for lesions composed of glandular cells with less atypia than AIS to define and characterize their association with other glandular processes. To avoid the circular argument of high-grade EGA (dysplasia) vs AIS, we set the minimum degree of AIS cells as the degree of atypia of the cells constituting a moderately differentiated invasive adenocarcinoma. Only 4 endocervical glandular lesions with mild atypia were found, 3 in patients with AIS or invasive endocervical-primary adenocarcinoma and 1 in a patient with invasive endometrial-primary, adenocarcinoma with endocervical extension. There were no lesions with high-grade atypia, nor was there a morphologic spectrum of cells with less atypia than AIS. Of the specimens, 14% had benign endocervical cell changes. The percentage of specimens in each group with benign endocervical cell changes was approximately equal. Although our study is small and retrospective, it suggests that no morphologic evidence exists to support the existence of a spectrum of endocervical glandular changes that culminates in AIS.

Cytologic diagnosis of peritoneal fluids. Applicability to the laparoscopic diagnosis of endometriosis.

OBJECTIVE: To determine the accuracy of cytologic examination of laparoscopically obtained peritoneal fluid in the diagnosis of endometriosis. STUDY DESIGN: This investigation analyzed 50 laparoscopic fluid specimens received over a three-year period. Retrospective cytologic findings were correlated with clinical history and laparoscopic diagnoses. Touch preparations were also collected from necropsies to develop cytologic criteria necessary to distinguish endometrial cells from mesothelial cells. RESULTS: The presence of hemosiderin-laden macrophages in peritoneal fluids was more specific but less sensitive than the presence of endometrial cells for the diagnosis of endometriosis. CONCLUSIONS: In women undergoing laparoscopy to detect endometriosis, the identification of endometrial cells alone in peritoneal fluids may not be sufficient to render a definitive diagnosis of endometriosis. However, the presence of hemosiderin-laden macrophages in this population should alert the cytologist to that possibility.

Characterization of human aortic smooth muscle cells expressing HPV16 E6 and E7 open reading frames.

A comparative study of human papillomavirus type 16 E6E7-transfected and normal human aortic smooth muscle cells by morphological, electron microscopic, immunofluorescent, and biochemical analyses demonstrated that the E6E7-expressing cells retained much of the phenotype of normal aortic smooth muscle cells, including expression of smooth muscle markers and appropriate growth responses to PDGF and heparin. These cells differed from normal vascular smooth muscle cells in that they had slightly altered morphology and a higher growth rate that was not due to an autocrine response to secreted PDGF, and they contained more polyribosomes than normal smooth muscle cells.

Breast fine-needle aspiration. A comparison of thin-layer and conventional preparation.

Two hundred forty-two breast fine-needle aspirates prepared by the Cytyc ThinPrep Processor were compared with aspirates prepared by the conventional smear method. Palpable and nonpalpable mammographic breast lesions were aspirated and the first half of the aspirate was submitted for conventional smears and the second half was rinsed into a proprietary fixative and loaded on the ThinPrep Processor for monolayer slide preparation. The matched pairs were diagnosed and analyzed separately in a double-blinded manner and later paired for comparison. Diagnoses correlated exactly in 62% of cases. The diagnosis of fibroadenoma was made in only 4 of 21 cases on ThinPrep (19% correlation). Semiquantitative analysis of several cytologic features indicated potential pitfalls for accurate diagnosis using the ThinPrep Processor. These included loss of background constituents (such as stroma and adipose tissue), decreased cellularity and single ductal epithelial cells, and decreased cytologic detail including size, shape and nuclear texture. The ThinPrep Processor may play a role in breast fine-needle aspiration, but further investigation is warranted before it is used as a sole preparatory method.

Calcium-activated K+ channels as modulators of human myometrial contractile activity.

The role of Ca(2+)-activated potassium (KCa) channels in the regulation of membrane potential, intracellular free calcium ([Ca2+]i) and contraction was investigated in uterine smooth muscle and myometrial cells. In an immortalized human myometrial cell line, oxytocin increased [Ca2+]i and [3H]inositol phosphate formation. Relaxin attenuated the oxytocin-induced increase in [Ca2+]i. In cell-attached patches, membrane depolarization activated a large-conductance KCa channel (179 +/- 4 pS). Iberiotoxin (IbTX), a potent blocker of "maxi" KCa channels (A. Galvez, G. Gimenez-Gallego, J. P. Reuben, L. Roy-Contanciin, P. Feigenbaum, G. J. Kaczorowski, and M. L. Garcia. J. Biol. Chem. 265: 11083-11090, 1990) produced long closed events (approximately 6 min) in these channels. In agreement with this blockage, IbTX depolarized the cells by 9.8 +/- 2.8 mV and caused a dose-dependent increase in [Ca2+]i with a half-maximal effective concentration of 0.79 nM. IbTX also caused phasic contractions in human myometrial strips and increased both the frequency and force of spontaneous contractions in estrogen-primed rat myometrial strips. Moreover, myometrial contractility was also affected by 1 mM tetraethylammonium, a concentration that blocks uterine smooth muscle KCa channels when applied to the extracellular side (G. J. Perez, L. Toro, S. D. Erulkar, and E. Stefani. Am. J. Obstet. Gynecol. 168: 652-660, 1993). These results strongly suggest that the large conductance KCa channels may actively participate in the control of human myometrial cell membrane potential and [Ca2+].

Interaction of HPV-18 and nitrosomethylurea in the induction of squamous cell carcinoma.

A strong correlation exists between the presence of specific types of human papillomavirus (HPV) and the development of anogenital cancer, as well as significant epidemiologic evidence suggesting smokers are at increased risk of developing cervical, vulvar and/or anal carcinomas. Primary and human papillomavirus type 18 (HPV-8)-immortalized human keratinocytes were used to address the co-carcinogenic potential of HPV and nitrosomethylurea (NMU) in tumorigenesis. Only cells containing HPV-18 and treated with NMU and the tumor-promoting phorbol ester, TPA, were transformed to a malignant phenotype. An in vitro system is described which initiates studies involving the mechanisms of HPV and chemical carcinogen co-operation in the etiology of squamous cell carcinomas.

Immortalization of primary human smooth muscle cells.

Primary human aortic and myometrial smooth muscle cells (SMCs) were immortalized using an amphotropic recombinant retroviral construct containing the E6 and E7 open reading frames (ORFs) of human papillomavirus type 16. The SMCs expressing the E6/E7 ORFs have considerably elevated growth rates when compared with nonimmortalized control cells and show no signs of senescence with long-term passage. The first SMC line derived in this study has been maintained in continuous tissue culture for greater than 1 year (greater than 180 population doublings). The immortalized SMCs have decreased cell size and decreased content of muscle-specific alpha-actin filaments as determined by indirect immunofluorescence. Southern blot analysis has demonstrated the stable integration of the E6/E7 ORFs in the retrovirally infected cells, and radioimmunoprecipitation has confirmed the continued expression of the E6 and E7 genes. Cytogenetic studies of the SMC lines have revealed essentially diploid populations except for the myometrial clonal line, which became aneuploid at late passage (greater than 125 doublings). These cell lines were not tumorigenic in nude mice.

Epithelial cells immortalized by human papillomaviruses have premalignant characteristics in organotypic culture.

Three HPV-16--and four HPV-18--immortalized human foreskin keratinocyte cell lines were analyzed on organotypic epidermal raft cultures at various passage levels. This culture system allowed normal cultured keratinocytes to stratify and differentiate in a manner similar to normal epidermis. All seven HPV-immortalized cell lines displayed epidermal morphologies on organotypic cultures, which were clearly abnormal and resembled premalignant lesions in vivo. Features of premalignant lesions that were shared by all of the HPV-immortalized cell lines included disorganized tissue architecture, mitotic cells present throughout the living layers of the epidermal sheet, abnormal mitoses, enlarged nuclei, and variable cell size and shape. Most HPV-immortalized cell lines were stable in terms of epidermal morphology with long-term passage in culture. Two of the HPV-18--immortalized cell lines, however, lost all morphologically apparent terminal squamous differentiation potential after long-term passage in monolayer culture. These results strongly support the idea that immortalization of squamous epithelial cells in culture by HPV-transforming genes generates a morphologically premalignant cell.

Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype.

We have developed a model system for progression of human epithelial cells to malignancy, using a human papillomavirus type 18 (HPV-18)-immortalized human keratinocyte cell line. Cells of cell line FEP-1811 were nontumorigenic in athymic mice through at least 12 passages in culture, but after 32 passages were weakly tumorigenic, producing tumors that regressed. After 62 passages they produced invasive squamous cell carcinomas that grew progressively. The progression to malignancy was associated with an increase in the efficiency of forming colonies in soft agar and with altered differentiation properties. In an organotypic culture system, FEP-1811 cells at passages 12 and 32 exhibited features typical of premalignant intraepithelial neoplasia in vivo, and cells at passage 68 exhibited features consistent with squamous cell carcinomas. No change in copy number of the transfected HPV-18 genome or in the level of expression of the viral transforming gene products E6 and E7 was detected between tumorigenic and nontumorigenic cells. Cytogenetic analysis of cells at early, middle, and late passage levels and cells cultured from tumors revealed that several chromosomal abnormalities segregated with the tumorigenic cell populations.

Thoracic disk herniation: MR imaging.

The authors undertook a retrospective study to assess the role of magnetic resonance (MR) imaging in thoracic disk herniation. The initial MR images were interpreted independently of other findings. These interpretations were compared with the findings of plain and computed tomography (CT) myelography and surgery, when available. Sixteen thoracic disk herniations were confirmed with plain and CT myelography and/or surgery. Plain myelography was performed on 14 patients and showed focal ventral filling defects in nine. Results of CT myelography were equivalent to those of MR imaging with three pulse sequences (sagittal T1 and T2 weighted, axial T1 weighted) in the identification of all the abnormal levels. In two patients, the signal from the herniated disk was so low on all sequences that thoracic disk herniation had to be inferred from the mass effect on the spinal cord. Precise location of the abnormal level with body coil MR images was achieved in six of 13 patients.

Angioinvasion in breast carcinoma. An immunohistochemical study of factor VIII-related antigen.

Histologic sections from 63 patients with infiltrating duct carcinoma of the breast were selected for study by immunohistochemical staining with antibody against human Factor VIII-related antigen. Of these 63, 30 had no lymph node metastases at the time of surgery, while 33 had axillary lymph node metastases. A positive correlation exists between the presence of vascular invasion and lymph node metastases. Sixty-nine percent of patients with lymph node metastases had vascular invasion while only 26% of patients without lymph node metastases showed evidence of invasion of blood vessels. The finding of vascular invasion by tumor in patients without axillary lymph node metastases at the time of mastectomy may explain the occurrence of disseminated disease years after treatment.

Squamous cell carcinoma of head and neck in patients with well-differentiated lymphocytic lymphoma.

Seventy-six cases of well-differentiated lymphocytic lymphoma (WDLL) were reviewed for evidence of additional malignancies. Of these, 18 patients (24%) had one to three further tumors; one half (nine) had squamous cell carcinoma (SCC). The head and neck area was the primary site in all but one case of SCC. The carcinomas were frequently multiple, recurrent, and clinically aggressive. The tumors showed unusually poor differentiation histologically. Actinic keratosis and basal cell carcinomas were frequently associated with SCC. Fifty percent metastasized to cervical lymph nodes replaced by WDLL. Lymphadenopathy due to metastatic SCC may be mistaken for malignant lymphoma alone. In two of five patients (40%), death was directly attributable to SCC. As in renal transplant recipients, SCC of the head and neck in WDLL patients is a common cause of significant complications and mortality requiring aggressive management. Immunosuppression due to WDLL and/or to chemotherapy is likely an important predisposing factor in combination with sun exposure.