奶山羊乳腺上皮细胞的分离、培养及鉴定

应用组织块培养法高密度培养、连续传代法建立西农萨能奶山羊乳腺上皮细胞体外培养体系,通过生长曲线绘制、核型分析、免疫荧光染色 (角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白)、油红染色及β-酪蛋白基因的RT-PCR分析进行培养细胞鉴定。实验结果表明细胞生长曲线为典型的S型,染色体数目众数为60,细胞角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白表达均呈阳性,油红染色后可见细胞质内的脂滴,且细胞表达酪蛋白mRNA。说明运用本方法培养的细胞为正常的乳腺上皮细胞,并具有一定的泌乳功能。     

Immunoperoxidase staining of cervicovaginal smears after radiotherapy.

Cervicovaginal smears from 2 women with postirradiation dysplasia, 4 women with postirradiation squamous cell carcinoma of the cervix, 30 women with irradiation atypia and 5 healthy, nonirradiated women were stained immunohistochemically with six keratin antibodies. For four of the antibodies--CK19 (BA17), EMA, PKK-1 and CAM 5.2--squamous cells showing irradiation atypia, postirradiation dysplasia or postirradiation squamous cell carcinoma were more likely to stain positively than were nonirradiated squamous cells. For three of the antibodies in which multiple squamous cells stained positively, the proportion of squamous cells showing postirradiation dysplasia or postirradiation squamous cell carcinoma staining strongly was equal to or greater than the corresponding overall proportion for squamous cells showing irradiation atypia. This was statistically significant with only one antibody, PKK-1. No statistically significant differences were seen in staining of irradiated and nonirradiated squamous cells by MAK-6 and AE1:AE3. The data show that some keratin antigens are more often expressed in the irradiated groups and that there may be differences in the degree of antigen expression between squamous cells showing postirradiation dysplasia or postirradiation squamous cell carcinoma and squamous cells showing irradiation atypia.     

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis

Studies were conducted to determine the effects of the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured human epidermal cells for comparison with known effects on mouse keratinocytes. In contrast to its effect on mouse cells, TPA did not stimulate human epidermal cell DNA synthesis. TPA stimulated differentiation in human keratinocytes resulting in sloughing of many cells by the 3rd day after exposure. Quantitative assays revealed that 50% of the TPA-exposed population was composed of cornified cells as opposed to 8% in untreated controls. A morphologically distinct cell type (TT cell) emerged after TPA treatment which was triangular in shape, did not stratify, appeared to proliferate rapidly and at most TPA concentrations became the predominant cell type within 1–2 weeks. Cultures composed predominantly of TT cells formed few cornified envelopes, grew well in the absence of TPA and formed colonies at low cell input. In contrast to its effect on keratinocytes, TPA enhanced TT colony formation 3–4-fold and decreased the doubling time of TT cells. Studies were performed to determine the origin of TT cells. Immunofluorescent staining indicated that TT cells lacked the keratinocyte antigens keratin, pemphigus and pemphigoid. Tonofilaments and desmosomes were not seen by electron microscopy. The lack of both melanosomes and standard histochemical DOPA oxidase staining indicated that TT cells were probably not of melanocyte origin. Tests used to identify Langerhans cells were negative. Whereas TT cells, as well as dermal fibroblasts, yielded positive immunofluorescence with antibodies to vimentin, TT cells gave a weak histochemical leucine aminopeptidase reaction, while the reaction of fibroblasts exposed to TPA was strong. Treatment of human dermal fibroblasts with TPA did not yield TT cells. The endothelial cell antigen factor VIII-associated protein was absent by immunofluorescence. These results suggest that the primary effect of TPA on cultured human epidermis is to accelerate terminal differentiation in the keratinocyte population and to stimulate growth of an as yet unidentified cell type.     

Neuroendocrine small cell carcinoma of the cervix associated with endocervical adenocarcinoma: a case report

BACKGROUND: Small-cell carcinoma (SCC) of the cervix is an uncommon member of the neuroendocrine group of cervical carcinomas that is frequently intermixed with a non-SCC component in the form of an adenocarcinoma (ADC) or squamous carcinoma. CASE: Colposcopy revealed a cervical mass in a 41-year-old woman and a Pap smear the presence of some tumor cells from SCC, which was confirmed by subsequent biopsy. The patient received 3 cycles of chemotherapy and then underwent major surgery. The cervical samples showed areas of endocervical ADC adjacent to and intermixed with the SCC. Reviewing the Pap smear, a previously missed malignancy was recognized. On subsequent molecular investigation to assess clonality by microsatellite analysis, the presence of HR-HPV DNA18 on real-time polymerase chain reaction, p16(INK4a) fluorescence in situ hybridization status and the corresponding immunohistochemical expression supported the hypothesis that the two components of the tumor shared the same cell origin. CONCLUSION: SCC of the cervix is a rare but distinct HR-HPV-18-related cervical carcinoma often intermixed with a clonally related non-small cell component consisting of an ADC or squamous carcinoma. The presence of SCC tumor cells in a cervical smear should prompt a search for malignant glandular or squamous tumor cells.     

K562 erythroleukemia cells express cytokeratins 8, 18, and 19 and epithelial membrane antigen that disappear after induced differentiation.

The effects of differentiation-modulating drugs were studied on the expression of intermediate filaments (IFs) in the human K562 erythroleukemic cell line. The untreated cells contained typical cytoplasmic coiling bundles, positive for both vimentin and cytokeratin as judged by indirect immunofluorescence microscopy with monoclonal antibodies (Mabs). Some of the cells also showed bright immunoreactivity for epithelial membrane antigen (EMA), as revealed with a Mab and polyclonal antiserum. When exposed to hemin or to sodium butyrate, most of the cells became cytokeratin negative within 3 days and showed dispersion of vimentin fibrils. Upon exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the amount of both vimentin and cytokeratin appeared to be greatly increased within 3 days and was found both in dispersed cytoplasmic fibrils, in large spherical, eccentric aggregates, as well as in cytoplasmic fibrils in cells spreading on fibronectin. TPA induced a complete loss of proliferation, as judged by immunostaining with the Mab Ki-67. The effects of TPA were found to be irreversible and could be induced by only a short exposure to the drug. Western blotting analysis and monoclonal antibodies to individual cytokeratins revealed that untreated K562 cells expressed Mr 52,000 (No. 8), 46,000 (No. 18), and 40,000 (No. 19) cytokeratin polypeptides, which disappeared when the cells were exposed to hemin or to sodium butyrate to induce erythroid differentiation but were greatly enhanced when exposed to TPA. The monoclonal anti EMA antibody reacted in K562 cells with a single Mr 320,000 polypeptide that was also revealed in MCF-7 breast carcinoma cells. Human bone marrow cells or other leukemic cell lines with erythroid differentiation capacity (HEL and KG-1) did not contain cytokeratin- or EMA-immunoreactive cells, suggesting that in K562 cells these properties may rather represent abnormal cytodifferentiation or retrodifferentiation toward early embryonic mesenchymal cells, than a more general expression of epithelial features in human leukemic cells.     

Tissue polypeptide antigen (TPA) is related to the non-epidermal keratins 8, 18 and 19 typical of simple and non-squamous epithelia: re-evaluation of a human tumor marker

Because of the broad clinical interest which tissue polypeptide antigen (TPA) has attracted as a tumor marker, human cell lines and human tissues have been analyzed for TPA expression using immunofluorescence microscopy. Epithelial cell lines including HeLa, MCF-7, and A-431 are recognized by TPA antibodies whereas human lines of non-epithelial origin are not. The positive staining patterns coincide with keratin-type intermediate filaments of the cytoskeleton. On tissue sections a subset of epithelial cells including uterine epithelium, bile duct cells in liver and tumor cells in breast carcinoma are strongly positive; cells of the squamous epithelia of skin and tongue as well as cells of non-epithelial origin are negative. In immunoblots of human epidermis, human tongue mucosa, human hair follicles, Detroit 562 cells, HeLa cells, MCF-7 and RT-4 cells, only keratins 8, 18 and 19 show TPA antigenicity. Conversely a TPA preparation is recognized by various antibodies known to react with keratins, including alpha-IFA, KG 8.13.2 and two antibodies which recognize keratins 18 (CK2) and 19, respectively. Our results thus relate TPA to human keratins 8, 18 and 19 which are known cytoskeletal components in both normal and malignant epithelial cells of simple and non-squamous origin. We speculate that the elevated levels of circulating TPA antigenicity present in the sera of patients with carcinoma, which are often used to monitor tumor progression, correspond to soluble proteolytic fragments originating from this particular keratin subgroup.     

胎盘X细胞的免疫组织化学研究

胎盘Ｘ细胞为一种绒毛外滋养层细胞,作者对２５例晚期妊娠胎盘中Ｘ细胞进行了Ｋｅｒａｔｉｎ、ＥＭＡ、ＨＣＧ、ＨＰＬ、ＰＬＡＰ、Ｐｒｏｌａｃｔｉｎ等免疫组织化学研究,其中Ｋｅｒａｔｉｎ、ＥＭＡ、ＨＰＬ、ＰＬＡＰ等染色均示部分细胞呈阳性反应。上述结果表明Ｘ细胞介于合体滋养细胞与细胞滋养细胞之间,符合中间滋养细胞的光镜及免疫组织化学特征,而与蜕膜细胞完全不同。     

Characterization of subcolumnar reserve cells and other epithelia of human uterine cervix. Demonstration of diverse cytokeratin polypeptides in reserve cells

We have analyzed the expression of cytokeratin polypeptides in subcolumnar reserve cells of the human uterine endocervical mucosa and the other epithelial cells using immunoperoxidase and immunofluorescence microscopy as well as by applying two-dimensional gel electrophoresis to microdissected cytoskeletal preparations. Endocervical columnar cells were uniformly positive for antibodies directed against the simple epithelium-type cytokeratins nos. 7, 8, 18, and 19, while a variable proportion of these cells was stained by an antibody against cytokeratin no. 4. Reserve cells were not only positive for cytokeratins nos. 8 (weakly and variably) and 19 but were also decorated by antibody KA 1, which reacts with cytokeratins present in stratified squamous epithelia. This last antibody selectively decorated reserve cells even when they were flat and inconspicuous. Antibody KA 1 uniformly stained the ectocervical squamous epithelium, the basal cells of which were also decorated by antibodies directed against cytokeratins nos. 8 (weakly and variably) and 19. Ectocervical suprabasal cells were positive, to a variable extent, for antibodies against cytokeratins nos. 4, 10/11, and 13. Gel electrophoresis revealed the presence of squamous-type cytokeratins nos. 5 and 17 in reserve cell-rich, but not in reserve cell-free, endocervical mucosa. We also analyzed the distribution pattern of these cells, as revealed by antibody KA 1, in the endocervical mucosa of 26 uteri. In all the specimens examined reserve cells were present, but their numbers exhibited considerable variation. In some cases these cells were confined to small islets localized deep within the cervical canal and lacked any continuity with the squamous epithelium. The expression of cytokeratins nos. 5 and 17 in reserve cells indicates that these cells have undergone a low level of squamous differentiation. The additional expression of cytokeratins nos. 8 and 19 in these cells points to a relationship with simple epithelial cells. The present data would seem to favor the view that reserve cells originate in situ from the columnar epithelium; however, this would imply an acquisition of new differentiation properties.     

抗人尿道高分化鳞癌单克隆抗体的研制

用本室建立的人尿道高分化鳞状上皮癌细胞株(Hus-98)免疫Balb/C小鼠,通过细胞杂交技术及ELISA方法进行筛选,SP免疫组织化学方法进行鉴定,获得10株能稳定分泌、选择性强、效价高的杂交瘤细胞株;同时从乳腺癌组织中提出肿瘤相关抗原行免疫印记实验(Western-blot)。结果6株经4次克隆能稳定分泌抗体Ig,免疫组化显示这些抗体是针对来源于上皮的鳞癌、腺癌细胞膜和细胞浆的,而癌旁正常组织及胎儿组织呈阴性反应;通过western-blot结果显示肿瘤相关抗原分子量为46.414kb。     

Evidence for a role of the monoclonal antibody E48 defined antigen in cell-cell adhesion in squamous epithelia and head and neck squamous cell carcinoma

Monoclonal antibody (MAb) E48 recognizes a 20- to 22-kDa antigen expressed by human squamous and transitional epithelia and their neoplastic counterparts. Histochemical examination of these tissues revealed distinct surface labeling with MAb E48. To investigate the subcellular localization of the E48 antigen we have performed electron microscopical analysis. In cells of normal oral mucosa, the E48 antigen was expressed on the plasmalemma, particularly associated with desmosomes, suggesting involvement of the E48 antigen in intercellular adhesion. Furthermore, the level of expression of the E48 antigen appeared to be influenced by the cellular organization. In squamous cell carcinoma (SCC) cell lines grown in vitro as subconfluent monolayer cultures, the E48 antigen expression was low. However, E48 antigen expression increased when SCC cells were grown to confluency. E48 antigen expression was similarly high when SCC cell lines were cultured under conditions promoting three-dimensional growth either as colonies within floating collagen gels or as xenograft in tumor-bearing nude mice. Further evidence for the involvement of the E48 antigen in cell-cell adhesion was found when SCC cells were grown within collagen gels in the presence of MAb E48: no spherical colonies were formed, but cells grew out to colonies composed of single cells. Moreover, in this culture system the percentage of SCC cells growing out to colonies was decreased by the presence of MAb E48. These findings indicate that the E48 antigen is involved in the structural organization of squamous tissue and might have a role in intercellular adhesion.     

Establishment and characterization of a new SCC antigen producing cell line (HCS-2) from a carcinoma of the uterine cervix

The cell line designated HCS-2 established from a squamous cell carcinoma of the uterine cervix has been subcultivated 77 times since Nov. 8, 1983. The cultured cells appear epithelial in shape, with a pavement-like arrangement and grow without contact inhibition. In electron microscopy, the cells are characterized by desmosomal cell contacts and a few tonofilaments. The cells are transplanted subcutaneously to nude mice and produce tumor which resembles the original tumor of large cell non-keratinizing squamous cell carcinoma. The growth rate of subculture has increased gradually, and population doubling time of cells at 17th passage was about 65 hours. The chromosome studies show aneuploidy and chromosomal number was mainly from hypertriploid to hypotetraploid range. The modal number of cells at 33rd passage was 81. Specific marker chromosome is not realized. The production of SCC antigen is detected from the cells and the amount of SCC antigen in cultured media was recorded from 1.5 to 2.0 ng per 1 x 10(4) cells for 48 hours. CEA synthesis is also confirmed immunohistochemically.     

Early expression of vimenti in human mammary cultures

Summary The intermediate filaments of most epithelial cells in vivo consist solely of cytokeratins. Using monoclonal antibodies to vimentin or keratin, we have examined the expression of vimentin in homologous specimens of frozen tissue sections and primary cultures of normal human mammary epithelium. In frozen sections, only epithelial cells reacted with the antikeratin antibody, whereas antivimentin reactivity was associated with stromal cells. All epithelial cultures were positive for cytokeratin and in addition coexpressed vimentin as strongly as cultured fibroblasts and as early as the 4th d after initiation of the culture. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of cytoskeletal preparations of secondary cultures of normal mammary epithelium have also demonstrated the appearance of a moiety identical to the vimentin found in cultured fibroblasts. Our observations are consistent with the hypothesis that vimentin expression is induced, possibly as a result of changes in cell shape or growth rate, when cells are freed from three-dimensional restirctions imposed by the tissue of origin     

Characterization of an HPV-negative cell line (FR-car) derived from a cervical squamous intraepithelial lesion

Summary 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.     

Preoperative CEA, NSE, SCC, TPA and CYFRA 21.1 serum levels as prognostic indicators in resected non-small cell lung cancer.

In 62 patients affected by resectable non-small cell lung cancer (NSCLC) submitted to radical surgery we evaluated the prognostic significance of CEA, NSE, SCC, TPA, and CYFRA 21.1 serum levels at diagnosis, as well as the predictive ability of these tumor markers with respect to histological type and pathological stage. The group was composed of 56 male and 6 female patients; the median age was 62 years (range 29-73 years). Thirty-four patients had a histological diagnosis of adenocarcinoma and 28 of squamous cell carcinoma; with regard to pathological stage, 32 patients had stage 1, 4 patients stage II and 23 patients stage IIIA disease. A good predictive ability with respect to histological type was obtained with SCC serum levels; as for pathological stage, TPA and CYFRA 21.1 were found to have moderate predictive ability. In this series of patients, at a median follow-up of 55 months after surgery, we found that both TPA and CYFRA 21.1 serum levels at diagnosis were reliable predictors of overall survival, high values of these markers being associated with a worse prognosis.     

Distinction between carcinoma cells and mesothelial cells in serous effusions. Usefulness of immunohistochemistry

The usefulness of an immunoperoxidase battery to distinguish carcinomatous from benign effusions was examined. Cell block sections from 90 previously diagnosed effusions were stained with antibodies to Leu-M1, B72.3, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA) and vimentin. The 90 cases comprised 69 carcinomas (23 mammary, 16 ovarian, 10 pulmonary, 7 gastrointestinal [GI] and 13 others), 2 malignant mesotheliomas and 19 cases with reactive mesothelial cells only. EMA and vimentin were the most useful markers for distinguishing carcinoma cells from reactive mesothelial cells. EMA reacted with 86% of the carcinomas while vimentin reacted with 90% of the reactive cases. Leu-M1, B72.3 and CEA, although generally less sensitive than EMA, were also helpful in this regard. Additionally, the use of Leu-M1 and CEA together may help to distinguish pulmonary from GI carcinomas.     

IMP-3 expression in keratoacanthomas and squamous cell carcinomas of the skin: an immunohistochemical study

The protein insulin-like growth factor II mRNA binding protein 3 (IMP-3) is an important factor for cell migration and adhesion in malignancies. Recent studies have shown a remarkable overexpression of IMP-3 in different human malignant neoplasms and also revealed it as an important prognostic marker in some tumor entities. The purpose of this study is to compare IMP-3 immunostaining in cutaneous squamous cell tumors and determine whether IMP-3 can aid in the differential diagnosis of these lesions. To our knowledge, IMP-3 expression has not been investigated in skin squamous cell proliferations thus far. Immunohi-stochemical staining for IMP-3 was performed on slides organized by samples from 67 patients, 34 with keratoacanthoma (KA) and 33 with primary cutaneous squamous cell carcinoma (SCC) (16 invasive and 17 in situ). Seventyfour percent of KAs (25/34) were negative for IMP-3 staining, while 57% of SCCs (19/33) were positive for IMP-3 staining. The percentage of IMP-3 positive cells increased significantly in the invasive SCC group (P=0.0111), and particularly in the SCC in situ group (P=0.0021) with respect to the KA group. IMP-3 intensity staining was significantly higher in invasive SCCs (P=0.0213), and particularly in SCCs in situ (P=0.008) with respect to KA. Our data show that IMP-3 expression is different in keratoacanthoma with respect to squamous cell carcinoma. IMP-3 assessment and staining pattern, together with a careful histological study, can be useful in the differential diagnosis between KA e SCC.Key words: IGF-II mRNA-binding protein-3 (IMP-3), keratoacanthoma, squamous cell carcinoma.     

Antibodies to intermediate filament proteins in the immunohistochemical identification of human tumours: an overview

Intermediate-sized filament proteins (IFP) are tissue specific in that antibodies to keratin, vimentin, desmin, glial fibrillary acidic protein (GFAP) and the neurofilament proteins can distinguish between cells of epithelial and mesenchymal origin as well as of myogenic and neural origin respectively. Malignant cells retain their tissue-specific IFP, which makes it possible to use these antibodies in tumour diagnosis. Carcinomas are exclusively detected by antibodies to keratin. Monoclonal antibodies to keratin have allowed the differentiation between subgroups of epithelial tumours until now between adenocarcinomas and squamous cell carcinomas. Lymphomas, melanomas and several soft tissue tumours are distinctly recognized by antibodies to vimentin. On the other hand, rhabdomyosarcomas and leiomyosarcomas are positive for desmin, while astrocytomas give a strong reaction with GFAP antibodies. Thus, antibodies to IFP are useful tools for differential diagnosis in surgical pathology.     

Differentiation of normal and tumoral human keratinocytes cultured on dermis: Reconstruction of either normal or tumoral architecture

Summary Normal human keratinocytes isolated from skin and squamous carcinoma cells established from a human tumor (TR146 cell line) both exhibit limited morphologic differentiation when they are grown on conventional plastic dishes. However, when they are seeded on human de-epidermized dermis and cultured at the air-liquid interface, they are able to reform an epithelium having the morphology of the tissue of origin (i.e. skin or squamous carcinoma). The distribution in such reconstructed tissues of differentiation markers such as bullous pemphigoid antigen, 67K keratin, involucrin, membrane-bound transglutaminase, and filaggrin was very similar to their distribution in normal skin and squamous carcinoma specimens, respectively. The degree of differentiation is for both cell types extremely sensitive to culture conditions such as retinoic acid concentration, emersion of the cultures, etc. These results show that subcultured normal or tumoral keratinocytes are able to recover their specific morphogenetic potential when cultured in an environment close to their in vivo situation.     

Immunohistochemical study of a case of malignant müllerian mixed tumor in comparison with the activity of normal uterine tissue

A case of malignant Müllerian mixed tumor of the uterus, exhibiting a histology of heterologous osteosarcomatous differentiation, is presented. Special emphasis is placed on the characteristic immunohistochemical reactivity of the tumor tissue in comparison with that of normal uterine tissue in the proliferative phase. Keratin, cytokeratin, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA), and human chorionic gonadotropin (HCG) were found only in the carcinomatous element. However, neuron specific enolase (NSE), S-100 protein (S 100) and vimentin were identified in almost all tumor tissue elements. Desmin and actin were not stained in any elements. Myoglobin was only detected weakly in the squamous carcinomatous element. Undifferentiated cell element, composed of small, round, spindle, or polygonal cells showed positive reactions to NSE and S 100, but not to any other antibodies. As compared to the reactivities of the normal proliferative endometrium, the glandular epithelial cells were positive with NSE, S 100, vimentin and CEA, but the stromal cells were only positive with vimentin. Such a multitudinous and concomitant expression of antigenicity to the different tumor elements indicates a close relationship to its mesodermal Müllerian origin, and NSE, S 100 and vimentin might be most adequate indicators of these types of tumors.     

Transforming Growth Factor β1Induces Squamous Carcinoma Cell Variants with Increased Metastatic Abilities and a Disorganized Cytoskeleton

Previous studies indicated that mouse transformed keratinocytes undergo an epithelial–fibroblastic conversion when cultured in the presence of TGF-β₁. This conversion is associatedin vivowith a squamous-spindle carcinoma transition. We derived epithelioid (A6, FPA6) and spindle (B5) clonal cell variants from a squamous carcinoma cell line (PDV) after treatment with TGF-β₁. FPA6 cells were isolated from the ascites fluid of an A6-tumor-bearing mouse. FPA6 and A6 cell lines produced in nude mice mixed carcinomas with a squamous and poorly differentiated component. Both cell lines coexpressed keratins and vimentin and synthesized E-cadherin protein, although FPA6 cells cultured at early passages (FPA6-ep) had reduced levels of E-cadherin mRNA and increased synthesis of keratin K8, a marker of malignant progression. Immunofluorescence analysis revealed that FPA6-ep cells exhibited a disorganized cytoskeleton with keratins forming focal juxtanuclear aggregates and loss of F-actin stress fibers and cortical bundles, and E-cadherin was localized in the cytoplasm out of cell–cell contact areas. Sporadic cells in A6 and PDV cultures also presented those anomalous keratin structures, suggesting that FPA6 cells originated from a subpopulation of A6 tumor cells that metastasized into the peritoneal cavity. The analysis of the spontaneous and experimental metastatic potentials of the cell lines showed that epithelioid and fibroblastic cell variants had acquired metastatic abilities compared to PDV which was nonmetastatic. The FPA6-ep cell line exhibited a highly aggressive behavior, killing the animals at about 17 days after intravenous injection of the cells into athymic mice. The phenotype of FPA6-ep cells was unstable and reverted at later passages in which the normal organization of keratin and F-actin in filaments and the localization of E-cadherin at cell–cell contacts were restored. This phenotypic reversion occurred concomitantly with a reduction of the experimental metastatic potential of FPA6 cells.