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C Paraskeva

Publications and source records attributed to C Paraskeva.

107 records · Page 6Linked to original sources
PubMed1988

Immortalization of a human colorectal adenoma cell line by continuous in vitro passage: possible involvement of chromosome 1 in tumour progression.

A non-tumorigenic epithelial cell line designated PC/AA, derived from a large pre-malignant colorectal adenoma from a patient with familial polyposis coli (also referred to as hereditary adenomatosis of the colon and rectum) has become immortal in vitro. PC/AA has been passaged in vitro continuously for over 4 years and shows no signs of senescence. At early passage, PC/AA has a normal diploid karyotype but with late passage is showing signs of progression, becoming aneuploid and displaying signs of morphological transformation. Every cell examined of late-passage PC/AA has an isochromosome (1q), and one other marker chromosome which is probably derived from an additional chromosome 8. The majority of cells examined have 48 chromosomes. Despite showing signs of progression in vitro, late-passage PC/AA has remained non-tumorigenic in athymic nude mice and retained morphological differentiation characteristics of colonic cells, in particular the ability to synthesize and secrete mucin. Two other cell lines derived from small adenomas did not become immortal in vitro and were also non-tumorigenic in athymic nude mice. The isolation of an immortal pre-malignant human epithelial cell line could prove invaluable in studies on human carcinogenesis and tumour progression. Our results, showing that only a large adenoma and no small adenomas have given rise to immortal cell lines, raise the possibility that the acquisition of in vitro immortality is associated with a relatively late stage in the adenoma-carcinoma sequence. The possible involvement of chromosome 1 in tumour progression is discussed.

Adenoma↗
PubMed1988

Expression of carcinoembryonic antigen by adenoma and carcinoma derived epithelial cell lines: possible marker of tumour progression and modulation of expression by sodium butyrate.

We have used an indirect immunofluorescence assay to demonstrate the cell membrane expression of carcinoembryonic antigen (CEA) by a pre-malignant colorectal adenoma derived epithelial cell line (PC/AA) and three colorectal carcinoma cell lines (HT29, PC/JW and PC/JW/FI). The results obtained indicated that CEA may be used as a marker for tumour progression up to the point of malignant transformation, after which the selection for anaplastic variants during continuous in vitro culture may result in the subsequent reduction of cell membrane CEA expression. The percentage of PC/AA cells expressing cell membrane CEA increased from 23.1% of diploid early passage (passage 18) cells to 56.0% of aneuploid late passage (passage 58) cells. Although non-tumorigenic, the proportion of PC/AA cells expressing cell membrane CEA at late passage corresponded to that for the PC/JW carcinoma line (56.2%) and is further evidence for the progression of PC/AA in culture. A 3T3 feeder-independent variant of PC/JW (PC/JW/FI) demonstrated a similar percentage of CEA-positive cells as the parental line for the first 21 passages without feeder support, but by passage 27 without 3T3 feeders only 35.3% of cells stained positive. This could be restored to 62.0% by continuous treatment with sodium butyrate (2 mM). A differential growth response to sodium butyrate was noted for the pre-malignant adenoma cell line PC/AA and the carcinoma lines HT29 and PC/JW/FI. Concentrations of sodium butyrate (2 mM) that killed early passage PC/AA cells allowed the late passage PC/AA cells and the carcinoma lines to proliferate, raising the possibility of sodium butyrate acting as a tumour promotor in the human colorectum.

Adenoma↗
PubMed1988

A study of ras gene mutations in colonic adenomas from familial polyposis coli patients.

Using an in vitro amplification step (polymerase chain reaction) followed by oligonucleotide dot blot analysis, DNA samples from 29 familial polyposis coli patients (75 polyp-derived and 26 'normal' colon samples with no epithelial atypia) were screened for the presence of K-, N-, and H-ras mutations. Only 5 polyps contained ras mutations (7%)--all in K-ras codon 12. In each case 'normal' colon DNA was available and found to be negative in this assay. We also report the detection of K-ras codon 12 mutations in a stably non-tumorigenic immortal adenoma-derived cell line, PC/AA, and in a tumorigenic colorectal carcinoma cell line, PC/JW. Both epithelial cell lines were derived from different FPC patients. An activated K-ras gene was also found in cell line S/AN, isolated from a sporadic villous adenoma. These results provide further evidence that there are common molecular events involved in sporadic and hereditary colorectal carcinogenesis and that K-ras mutations can precede the development of malignancy. To our knowledge PC/AA is the first reported example of a human cell line bearing a mutant ras gene that is not tumorigenic and shows that the presence of an activated ras gene even in an immortal human cell line is insufficient for malignancy.

Adenoma↗
PubMed1986

Elevated production of growth factor by human premalignant colon adenomas and a derived epithelial cell line.

Growth factor activity which stimulates anchorage-independent growth (AIG) in a rat fibroblast line, was detected in human premalignant adenoma tissue from familial polyposis coli colectomy specimens and in serum-free culture supernatant from an adenoma cell line PC/AA. The activity extracted from adenoma tissue was compared quantitatively in the AIG bioassay with extracts of normal mucosa from split thickness colorectal tissue. Adenoma tissue yielded three times the amount of acid-extractable protein g-1 wet wt and adenoma extracts consistently had significantly greater specific activity over a wide protein concentration range. Activity extracted from adenoma tissue and from the derived cell line PC/AA were compared qualitatively after fractionation by gel filtration. Both extracts showed almost identical profiles of biological activity after assay of individual fractions for AIG stimulation, suggesting that the factor(s) originates from the epithelial component of the adenoma tissue since PC/AA is a pure epithelial cell line. Activity eluted as two major peaks with apparent mol. wts of 9 kd and 20-25 kd (relative to standards) in both cases. This report demonstrates for the first time that elevated production of a growth factor may be an early change in the evolution of human colorectal cancer from small, premalignant adenomas.

Adenoma↗
PubMed1984

The isolation and characterization of colorectal epithelial cell lines at different stages in malignant transformation from familial polyposis coli patients.

The genetic disease familial polyposis coli (hereditary adenomatosis of the colon and rectum) provides an excellent model for the study of tumour progression in the large bowel. We have isolated and characterized four epithelial cell lines from colorectal tumours from polyposis coli patients. These cell lines are grown on collagen-coated Petri dishes in the presence of mouse 3T3 feeder cells in medium containing 20% foetal bovine serum. Of these cell lines three were isolated from premalignant adenomas and one from an adenocarcinoma. All four lines have a characteristic cuboidal epithelial morphology, and their epithelial origin was confirmed by positive staining with a monoclonal antibody which reacts specifically with the keratin filaments of simple epithelia. The adenoma-derived lines display ultrastructural features characteristic of colonic epithelium including desmosomes, microvilli and mucin droplets. One of the adenoma-derived cell lines, designated PC/AA, has retained differentiated functions in culture, namely mucin production, after 21 in vitro passages. PC/AA has a karyotype of 46, XY with no detectable chromosome rearrangements. The adenoma-derived lines could be passaged from clumps of cells but not from single cells even in the presence of 3T3 feeder cells. The carcinoma-derived line, designated PC/JW, could however grow from single cells in the presence of a feeder layer. The one premalignant adenoma-derived line tested so far, PC/AA, did not produce tumours in athymic nude mice. In contrast, the carcinoma-derived line, PC/JW was tumorigenic in athymic nude mice. PC/JW produced moderately well-differentiated tumours which were histologically similar to the adenocarcinoma from which the cell line was isolated. PC/JW has a near-diploid chromosome number with an isochromosome (1q), an isochromosome (14q) and an (Xp; 17q) translocation. Unidentified marker chromosomes were present in a few cells. The features at present which distinguish the carcinoma-derived line from the adenoma-derived lines are tumorigenicity, growth from single cells and chromosomal abnormalities. The isolation and characterization of differentiating human epithelial cell lines at different stages in malignant transformation provide an opportunity to examine the cellular and molecular mechanisms controlling tumour progression in the large intestine, and to obtain an insight into the multistep process of human epithelial carcinogenesis.

Adenoma↗
PubMed1983

Human adenovirus type 2 but not adenovirus type 12 is mutagenic at the hypoxanthine phosphoribosyltransferase locus of cloned rat liver epithelial cells.

Using resistance to the base analog 8-azaguanine as a genetic marker, we showed that adenovirus type 2, but not adenovirus type 12, is mutagenic at the hypoxanthine phosphoribosyltransferase locus of cloned diploid rat liver epithelial cells. Adenovirus type 2 increased the frequency of 8-azaguanine-resistant colonies by up to ninefold over the spontaneous frequency, depending on expression time and virus dose.

Adenoviruses, Human↗
PubMed1982

Adenovirus-cell interactions early after infection: in vitro characteristics and tumourigenicity of adenovirus type 2-transformed rat liver epithelial cells.

Cloned rat liver epithelial cells (clone C3) were semi-permissive for adenovirus type 2 (Ad-2) and non-permissive for adenovirus type 12 (Ad-12). Ad-2-infected C3 cells were shown to produce hexon and fibre protein, but at an m.o.i. of 20 a maximum virus yield of only 2.4 p.f.u. per cell was obtained. Forty-eight h after infection with Ad-12 'early' virus proteins (major species 8K and 60K), but no 'late' proteins (virus structural proteins) could be identified. Of six Ad-2-transformed epithelial lines isolated from clone C3 only one was tumourigenic in syngeneic rats, whereas all six transformants produced tumours in athymic nude mice. There was a remarkable variation in the morphology of the Ad-2-transformed liver cells, ranging from an epithelial morphology similar to C3 cells to cells with a distinct lymphoid morphology. The in vitro and in vivo behaviour of the Ad-2-transformed clone C3 cells reported in this communication, taken together with our previous report on the characteristics of Ad-12-transformed C3 cells, clearly show that the differences observed between Ad-2- and Ad-12-transformed rat embryo cells were also observed in our studies using cloned rat liver epithelial cultures. Our findings clearly rule out the hypothesis that the heterogeneity of Ad-2-transformation events is the result of the transformation in vitro of different types of target cell.

Adenoviruses, Human↗
PubMed1982

Adenovirus type 12-transformed rat embryo brain and rat liver epithelial cell lines: adenovirus type 12 genome content and viral protein expression.

By Southern blotting analysis, six adenovirus type 12 (Ad-12)-transformed rat embryo brain cell lines and one Ad-12-transformed rat liver epithelial line were shown to contain all or nearly all the Ad-12 genome. Another Ad-12 rat liver epithelial cell line contained a repeating structure consisting of only the left-hand 16% of the Ad-12 genome. Three Ad-12-specified proteins (molecular weights, 52,000, 41,000, and 18,000) were found by immunoprecipitation to be common to all of these cell lines. Two rat liver epithelial lines, produced from an Ad-12-infected culture and previously shown to be T-antigen negative by immunofluorescence, contained no detectable Ad-12 genome or Ad-12-specified proteins. Although some of the rat embryo brain transformants had been shown previously to express "late" Ad-12 mRNA, no Ad-12 structural proteins were found to be produced by these cell lines.

Adenoviruses, Human↗
PubMed1981

Sensitivity of normal and transformed rat liver epithelial cells to benzo[a]pyrene toxicity.

A strain of rat liver epithelial cells (LP), a clone of LP (C3) and a series of transformed lines derived from these cell strains, were examined for their sensitivity to the toxic effects of benzo[a]pyrene (BP). Both LP and C3 were sensitive to BP and within the series of transformed cell lines, sensitivity to BP was positively related to the extent of differentiation of the tumours each line formed in vivo. All the cell types were able to take up BP and there appeared to be a relationship between the sensitivity of a given cell type to BP, and the amount of BP it metabolised. The binding of BP to the DNA was low in all the cell types examined.

Animals↗
PubMed1980

Tumorigenicity and in vitro characteristics of rat liver epithelial cells and their adenovirus-transformed derivatives.

Cloned and uncloned epithelial cultures were established from the liver of a 3-week-old AS rat. These epithelial cultures were neither tumorigenic nor did they display anchorage-independent growth. One of the clones was cytogenetically normal after 53 in vitro passages (approximately 200 population doublings after cloning). Eight transformed lines were isolated from the liver epithelial cells after infection with adenovirus type 12 (Ad-12). Five of these produced typical Ad12 T-antigen, whereas three appeared to be T-antigen-negative. All were tumorigenic in newborn syngeneic rats. The T-antigen-positive transformed lines produced anaplastic-epithelial tumors, whereas the T-antigen-negative transformed lines produced adenocarcinomas. Although all the transformed lines were tumorigenic, some were fibronectin-positive while others produced no detectable fibronectin. The normal (untransformed) epithelial cells produced fibronectin. These results are interesting for two reasons: (1) there are relatively few reports of fibronectin on epithelial cells and (2) they emphasize the view that there is no absolute correlation between reduced fibronectin and tumorigenicity in transformed cells. The transformed lines displayed in vitro characteristics similar to those of transformants derived from embryonic and fibroblastic cell strains, notably, increased saturation density and changes in cellular morphology. Some of the transformed cell lines, but not all, displayed anchorage-independent growth. All the transformed cell lines were picked from multi-layered foci so that morphological criteria (i.e. piling-up focus) for isolating transformants from the epithelial cultures were similar as in embryonic and fibroblastic transforming cell systems. With the new cell system we have developed we can, using the same epithelial cell line (clone C3), study both virus transformation and virus mutagenesis.

Adenocarcinoma↗
PubMedDate not supplied

The protrusive phase and full development of integrin-dependent adhesions in colon epithelial cells require FAK- and ERK-mediated actin spike formation: deregulation in cancer cells.

Integrins play an important role in tumour progression by influencing cellular responses and matrix-dependent adhesion. However, the regulation of matrix-dependent adhesion assembly in epithelial cells is poorly understood. We have investigated the integrin and signalling requirements of cell-matrix adhesion assembly in colon carcinoma cells after plating on fibronectin. Adhesion assembly in these, and in the adenoma cells from which they were derived, was largely dependent on alpha v beta 6 integrin and required phosphorylation of FAK on tyrosine-397. The rate of fibronectin-induced adhesion assembly and the expression of both alpha v beta 6 integrin and FAK were increased during the adenoma-to-carcinoma transition. The matrix-dependent adhesion assembly process, particularly the final stages of complex protrusion that is required for optimal cell spreading, required the activity of extracellular signal-regulated kinase (ERK). Furthermore, phosphorylated ERK was targeted to newly forming cell--matrix adhesions in the carcinoma cells but not the adenoma cells, and inhibition of FAK--tyrosine-397 phosphorylation or MEK suppressed the appearance of phosphorylated ERK at peripheral sites. In addition, inhibition of MEK--ERK activation blocked the formation of peripheral actin microspikes that were necessary for the protrusive phase of cell-matrix adhesion assembly. Thus, MEK--ERK--dependent peripheral actin re-organization is required for the full development of integrin-induced adhesions and this pathway is stimulated in an in vitro model of colon cancer progression.

Actins↗
PubMedDate not supplied

Colorectal carcinogenesis: sequential steps in the in vitro immortalization and transformation of human colonic epithelial cells (review).

The development of colorectal cancer is an excellent example of the complex multistage nature of carcinogenesis and most colorectal cancers are thought to develop from adenomas. In this paper we have reviewed in vitro models developed in our laboratory for the study of human colorectal carcinogenesis. For these studies epithelial cell lines have been isolated from hereditary and sporadic colorectal adenomas representing different stages in tumour progression. Karyotypic analysis has shown specific abnormalities of chromosomes 1, 7, 14, 17, 18 and 22 to occur in these premalignant adenoma cell lines. The majority of cell cultures derived from small adenomas (less than 1 cm in diameter) senesced whereas the larger adenomas (greater than 2 cm in diameter) were more likely to give rise to immortal cell lines indicating that the acquisition of in vitro immortality occurs at a relatively late stage of colorectal carcinogenesis. Abnormalities of chromosome I have been implicated in tumour progression and in the in vitro immortalization of colorectal adenomas. Furthermore, several stages have been described in the transformation of an adenoma cell line PC/AA to a tumorigenic phenotype. Sodium butyrate and the potent carcinogen N-methyl-N-nitro-nitrosoguanidine (MNNG) were used in this transformation. Sodium butyrate is proposed to act as a possible promoter of colorectal carcinogenesis, and MNNG to cause the further genetic changes required for the conversion of the premalignant cells to a carcinoma. Markers to study the progression of an adenoma cell line to a tumorigenic phenotype in vitro include in vitro immortalization, aneuploidy, clonogenicity, resistance to the inhibitory effects of sodium butyrate, anchorage independent growth, ras gene activation, production of active proteinases and tumorigenicity in athymic nude mice. A role for a constitutively produced tumour promoter in colorectal carcinogenesis is discussed together with the possibility that different events are involved in the development of sporadic versus hereditary tumours due to the importance of the microenvironment in hereditary cancer. Our in vitro progression provides the first experimental evidence for the adenoma to carcinoma sequence and the cytogenetic evidence suggests that it is relevant to in vivo carcinogenesis.

Adenoma↗
PubMedDate not supplied

Differential growth response to oestrogen of premalignant and malignant colonic cell lines.

Epidemiological and experimental evidence support a role for sex steroid hormones in colorectal cancer. The aims of this study were to determine expression of oestrogen receptor in adenoma derived and carcinoma derived colonic epithelial cell lines, and to determine the in vitro effect of beta oestradiol on growth. Between 0.5-1.5 fmol/mg protein of oestrogen receptor was expressed in the colonic cell lines studied. mRNA for oestrogen receptor was also expressed. An adenoma derived cell line, AA/C1, demonstrated an increase in growth rate in response to beta oestradiol. The effect was density dependent. The cell line AA/C1/SB10, a chemically transformed derivative of AA/C1, did not respond to beta oestradiol. Adenoma derived and carcinoma derived colonic cell lines express oestrogen receptor. Oestrogen may have a trophic action in vivo on premalignant colonic epithelium.

Breast Neoplasms↗