Fibrinolysis in a lipid environment: modulation through release of free fatty acids.

BACKGROUND: Thrombolysis is conventionally regarded as dissolution of the fibrin matrix of thrombi by plasmin, but the structure of clots in vivo includes additional constituents (proteins, phospholipids) that modulate their solubilization. OBJECTIVE: We examined the presence of free fatty acids in thrombi and their effects on distinct stages of fibrinolysis (plasminogen activation, plasmin activity). METHODS AND RESULTS: Using the fluorescent probe acrylodated intestinal fatty acid-binding protein, variable quantities (up to millimolar concentrations) of free fatty acids were demonstrated in surgically removed human thrombi. Oleic acid at relevant concentrations reversibly inhibits more than 90% of the amidolytic activity of plasmin on a synthetic substrate (Spectrozyme PL), but only partially inhibits its fibrinolytic activity measured using turbidimetry. Chromogenic assays detecting the generated plasmin activity show that plasminogen activation by tissue-type plasminogen activator (t-PA) is completely blocked by oleic acid in the fluid phase, but is accelerated on a fibrin matrix. A recombinant derivative of t-PA (reteplase) develops higher fibrin specificity in the presence of oleic acid, because both the inhibition of plasminogen activation in free solution and its enhancement on fibrin template are stronger than with wild-type t-PA. CONCLUSION: Through the stimulation of plasminogen activation on a fibrin template and the inhibition of plasminogen activators and plasmin in the fluid phase, free fatty acids confine the action of fibrinolytic proteases to the site of clotting, where they partially oppose the thrombolytic barrier function of phospholipids.

Role of the basement membrane in tumor cell dormancy and cytotoxic resistance.

OBJECTIVES AND METHODS: Tumor dormancy and resistance to cytotoxic agents are key limiting events in the treatment of malignant diseases. To determine whether both are influenced by the extracellular milieu in which tumors reside, HT1080 human fibrosarcoma, MCF-7 breast carcinoma and OSCORT osteosarcoma cell proliferation, viability, apoptosis and cytoreductive-treatment-induced death were investigated in the presence or absence of extracellular matrix (ECM). RESULTS: ECM-adherent, but not plastic-adherent HT1080 cells formed a multicellular network accompanied by reduced proliferation and lowered DNA synthetic capacity. The number of cells in S-phase was dramatically reduced. Viable cells entered a state of dormancy reminiscent of that observed in the step of metastasis after extravasation, i.e. prior to the initiation of progressive growth. Such ECM-induced dormancy could be reversed by plating cells on plastic, but only after a 48-hour lag period. No difference was indicated in clonogenicity of HT1080 cells originated from plastic or ECM gel. However, the cells released from ECM gel showed significantly reduced migration ability. The resistance of anchored cells against cytotoxic damage was increased by ECM gel. Examination of cytoreductive treatment revealed that ECM adherence at the time of injury is partially protective, a property which was also moderately apparent when injured cells were transferred to the basement membrane. CONCLUSIONS: Taken together, these results suggest that the ECM plays a key role in tumor dormancy and cytotoxic resistance, both explorable at the molecular level using our in vitro model system.

Formaldehyde promotes and inhibits the proliferation of cultured tumour and endothelial cells.

Formaldehyde was applied in various doses (0.1-10.0 mM) to HT-29 human colon carcinoma and HUV-EC-C human endothelial cell cultures. Cell number, apoptotic and mitotic index as well as proportion of cells in S-phase was investigated by morphological methods and flow cytometry. Ten mM of formaldehyde caused high degree of cell damage and practically eradicated the cell cultures. One mM of formaldehyde enhanced apoptosis and reduced mitosis in both types of cell cultures, in a moderate manner. The low dose (0.1 mM) enhanced cell proliferation and decreased apoptotic activity of the cultured cells, the tumour cells appeared to be more sensitive. The possible role of this dose-dependent effect of formaldehyde in various pathological conditions, such as carcinogenesis and atherogenesis is discussed with emphasis on the eventual interaction between formaldehyde and hydrogen peroxide.

Anti-apoptotic and apoptotic action of (-)-deprenyl and its metabolites.

The mode of cytoprotective action of the monoamine oxydase B inhibitor (-)-deprenyl was studied using A-2058 human melanoma cells in culture. Serum deprivation caused apoptosis of the cultured cells, which could be decreased by administration of 10(-9) - 10(-13)M (-)-deprenyl. The known metabolites of (-)-deprenyl, (-)-desmethyl-deprenyl, (-)- and (+)-methylamphetamine failed to exert the same effect. The anti-apoptotic activity of (-)-deprenyl was prevented by the simultaneous application of the microsomal drug-metabolizing enzyme inhibitor SKF-525A. These results show that (-)-deprenyl needs metabolic conversion in order to be anti-apoptotic, but the effective metabolite is still unknown. On the other hand, higher dose (10(-13)M) of (-)-deprenyl, (-)-desmethyl-deprenyl, (-)- and (+)-methylamphetamine induced apoptosis in the non-serum-deprived A-2058 cell culture. SKF-525A did not prevent the apoptosis-inducing effect of (-)-deprenyl, which means that no metabolic changes are needed for this activity. High dose (10(-3)M) of (-)-deprenyl induced very high Caspase 3 activity in non-serum-deprived A-2058 cell culture, low doses (10(-9) - 10(-3) M) of (-)-deprenyl maintained Caspase 3 activity on control level in case of serum-deprivation.

Interdigitation index - a parameter for differentiating between young and older skin specimens.

BACKGROUND/AIMS: Quantitative morphometry developed rapidly during the last decade due to advances is computers and software. We wish to establish a simple baseline for the morphometric differences due to intrinsic ageing between young and old cohorts: the interdigitation index. It is an expression of the shape of the border between the epidermis and dermis. METHODS: We used volar forearm biopsies of women, since the volar forearm is usually not photodamaged. The biopsies were fixed in buffered formalin, embedded in paraffin and sectioned. Separate sections were stained by hematoxylin-eosin, by orcein and by dimethyl-methylene blue. We had seven female volunteers in each group; the young cohort had a mean age of 26.6 years, the older cohort 50.9 years. We chose a cohort that was just about postmenopausal, since in the future we wish to evaluate the effect of externally-applied agents on postmenopausal female skin and the earlier it is applied the better its chance of being effective. RESULTS: We found no difference between the young and older cohort with regard to epidermal thickness. We found a decrease of glycosaminoglycen (GAG) as measured by dimethyl-methylene blue staining. The results of the elastic staining by orcein, although in line with the reports in the literature, are not useful for evaluating the intrinsic ageing process, at least not by the simple percentage of area stained procedure. We introduced a new parameter: the interdigitation index. It is a simple measurement of the interdigitation in the epidermal-dermal junction, known to be diminished by age. This index was diminished by about 20% between the young and older cohort. CONCLUSIONS: Quantitative morphometry using simple epidermal and dermal measurements on biopsies of the volar forearm of women is suitable for following intrinsic ageing of the skin and offers a simple objective method for following the ageing process of the skin.

Modulation of 5-fluorouracil by 5-ethyl-2'-deoxyuridine on cell lines expressing different dihydropyrimidine dehydrogenase activities.

The purpose of the present study was to clarify the significance of the inhibition of dihydropyrimidine dehydrogenase (DPD) in the modulation of 5-fluorouracil (5-FU) action by 5-ethyl-2'-deoxyuridine (EUdR). Four human cell lines, which differed in their susceptibility to 5-FU and in their DPD activity, were selected as biological objects. Several other enzymes of pyrimidine metabolism, i.e. thymidylate synthase (TS), thymidine kinase (TK) and pyrimidine nucleoside phosphorylase (PNP), which might be involved in the 5-FU action were also studied to elucidate their potential role in the modulation of 5-FU cytotoxicity. Two out of the four cell lines, i.e. COLO1 and SW620, showed low (57 and 28 pmol/min/mg protein) and the other two cell lines, i.e. CAL51 and CAL33, showed high (235 and 184 pmol/min/mg protein) DPD activity, respectively. In our study, contrary to our expectation, no correlation between the DPD and TS activity of the cell lines and their 5-FU sensitivity could be observed. EUdR alone was cytotoxic only on CAL33 cells in a concentration below 1 mM (IC50=194 microM) which might be due to the high TK activity (857 pmol/min/mg protein) measured in this cell line, favoring the formation of the phosphorylated nucleotides EdUMP and EdUTP indispensable for the inhibition of TS and DNA polymerase, respectively. Surprisingly, although EUdR by metabolizing to EUra was able to reduce the high activity of DPD in CAL33 and CAL51 cells by 47 and 55%, respectively, no potentiation of the 5-FU action occurred on these cell lines. On the contrary, enhancement of the 5-FU cytotoxicity was demonstrated on COLO1 and SW620 cells with low DPD activity. Our findings suggest that the 5-FU modulatory action of EUdR may be directed on other molecular targets than DPD as well, i.e. the augmentation of TS inhibition by EdUMP as demonstrated on SW620 cells might be one of these mechanisms.

The antiproliferative action of a melphalan hexapeptide with collagenase-cleavable site.

PURPOSE: The objective of the present study was to examine the relevance of collagenase in the antitumor action of a melphalan peptide (MHP) with a collagenase-cleavable sequence. The question was addressed as to whether collagenase may act as an activator or a target in the antiproliferative mechanism of MHP. METHODS: Melphalan was inserted into peptides representing the sequence Pro-Gln-Gly-Ile-Ala.Gly of the collagenase-cleavable site in collagens. Changes in growth and collagenase IV activities of HT-1080, HT-29, HT-168, and MCF-7 cell cultures were investigated. RESULTS: The present investigations provide data indicating that Pro-Gln-Gly-Ile-Mel-Gly (melphalan hexapeptide, MHP) is a substrate for both bacterial and 72-kDa type IV collagenases and that in this way it can generate Ile-Mel-Gly (melphalan tripeptide, MTP) of higher cytotoxic potency. Indeed, the formation of MTP was detected in the conditioned medium of HT-1080, a collagenase IV-producing human fibrosarcoma. In a comparison of equimolar concentrations of melphalan and its two peptide derivatives (MHP and MTP), superior antiproliferative action of MTP was seen in HT-29, HT-1080, and HT-168 tumor cell cultures. However, the relatively modest cytostatic actions of MHP were increased when bacterial collagenase was added to the cell cultures. After melphalan treatment, reduced levels of both 92 and 72-kDa type IV collagenases were seen in the HT-1080 cell cultures. However, the reduction of collagenase activity and the cell counts did not run parallel in the MTP- or MHP-treated cultures; indeed, collagenase activity related to cell numbers showed an elevated level. CONCLUSIONS: As the conversion of MHP to the more toxic MTP was detected in the presence of collagenases, it is possible that collagenase-directed activation of prodrugs may be a promising approach for the development of more selective cytostatic drugs against malignant tumors with high collagenase activities.

Establishment and characterization of a new transplantable pancreatic cancer xenograft (PZX-5) in immunosuppressed mice.

CONCLUSION: A new, stable, transplantable human pancreatic cancer xenograft (PZX-5) model has been established in CBA immunosuppressed mice. BACKGROUND: Numerous human pancreatic carcinomas have been successfully transplanted into athymic nude mice. However, artificially immunosuppressed animals have rarely been used as recipients. Because this model system proved to be reliable for hosting many human malignancies at our institute, successive xenotransplantations of a ductal adenocarcinoma have been carried out. METHOD: Immunosuppression of CBA/CA mice was achieved by thymectomy, whole-body irradiation and bone-marrow reconstruction. Tumor fragments were subcutaneously implanted from a well/moderately differentiated ductal pancreatic adenocarcinoma and serially transplanted for more than 20 mo. The xenografted tumors were characterized using morphological, immunohistochemical, biochemical, and flow cytometric methods. RESULTS: During the serial transplantations, the neoplasm maintained its original morphological-pathobiological characteristics. It produced a large amount of mucin and expressed carcinoembryonic antigen (CEA). Neither the mitotic activity nor the degree of differentiation was altered, and CEA was permanently detected. Flow cytometric DNA analysis revealed an aneuploid pattern (DNA index 1.45+/-0.03), which has remained within the same range during xenograftings. The doubling time in an in vitro system proved to be 18 h. The human character has been well preserved even 9 mo posttransplantation, as was evidenced by LDH-isoenzyme electrophoresis. The results indicate that the thymectomized--whole-body irradiated--bone-marrow reconstructed immunosuppressed mice are also appropriate hosts for pancreatic cancer xenografts.

Studies on the proliferative activity of diethylnitrosamine-induced hepatocellular carcinomas in monkeys.

In this study, synthetic phase fractions (SPFs) determined by flow cytometry and AgNOR counts were analysed in benign liver lesions (regenerative nodules and adenomas), hepatocellular carcinomas (HCCs), and lung metastases of a monkey hepatocarcinogenesis model to find out if AgNOR counts and SPFs can discriminate between malignant and non-malignant liver lesions. The average per cent SPF values and the AgNOR counts were significantly (P = 0.001) increased in regenerative liver nodules (5.30 per cent; 4.96), adenomas (5.34 per cent; 3.46) and well-differentiated HCCs (6.75 per cent; 4.47), compared with the untreated control livers (3.18 per cent; 0.98), but the differences between these three groups were not significant. In the poorly differentiated HCC group, however, the average SPF value (9.60 per cent) and AgNOR count (7.14) were significantly higher than in any of the other liver lesions examined. A significant correlation was found between the SPF values and AgNOR counts on the one hand, and differentiation and cytological grade of the HCC samples on the other. The results of this study show that the SPF values and AgNOR counts are not reliable in differentiating between regenerating liver nodules, adenomas, and experimental well-differentiated HCCs. The SPF value, however, may serve as a prognostic factor in HCC, since it was found to be significantly higher in HCCs with lung metastasis than in those without.

Quantitative evaluation of lysozyme- and CD68-positive Kupffer cells in diethylnitrosamine-induced hepatocellular carcinomas in monkeys.

Quantitative analysis of lysozyme- and CD68-positive Kupffer cells was carried out in connection with diethylnitrosamine-induced hepatocarcinogenesis in non-human primates. The number of Kupffer cells/mm2 was determined in 28 cases of hepatocellular carcinoma (HCC) and seven age-matched controls. The Kupffer cell counts (mean +/-SEM) gradually decreased in the following order, irrespective of the histochemical markers (lysozyme or CD 68) used: healthy control liver (101.7 +/- 13.5 and 103.2 +/- 11.9 respectively), non-cirrhotic and non-neoplastic host liver (54.3 +/- 13.6 and 50.5 +/- 15.4), cirrhotic host liver (26.2 +/- 8.2 and 27.2 +/- 3.3), HCC tissue (20.7 +/- 4.4 and 19.3 +/- 4.1) and metastatic foci in the lung (9.8 +/- 1.8 and 9.7 +/- 2.8). The difference between the normal liver and the non-neoplastic, non-cirrhotic portions of the HCC-bearing liver was significant (P < 0.05). A highly significant difference was found between the number of Kupffer cells found in healthy control or non-neoplastic liver and those found in HCC nodules (P < 0.0001 and P < 0.0005 respectively). The results obtained by hematoxylin and eosin staining and lysozyme/CD68 immunohistochemistry were highly similar, indicating that this decrease was attributable primarily to numeric loss of Kupffer cells. The results suggest that the reduction in the number of Kupffer cells in HCC is a constant feature of hepatocarcinogenesis not only in rodent models, but also in non-human primates.

Olive oil decreases liver damage in rats caused by carbon tetrachloride (CCl4).

Oral carbon tetrachloride (CCl4) poisoning of the liver of male F-344 rats was modified by dissolving CCl4 in various oils (sunflower, corn, fish and olive). After 8 weeks of CCl4 treatment (3 x 0.2 ml/kg body weight every other day, dissolved in aliquots of 0.5 ml of each types of oil) the rats were sacrificed and the ratio of connective tissue in the liver was determined by morphometry after picrosirius staining. The percentage of collagen fibres increased in all CCl4-treated groups compared to the controls. This increase was almost the same (6-8%) in the case of CCl4 dissolved in sunflower, corn or fish oil, but when olive oil was applied as a solvent, the collagen ratio was only 2-4 percent. On the bases of this finding olive oil is considered as less harmful to the liver in acute CCl4 poisoning than other oils.

Role of the modified (glycosaminoglycan producing) perisinusoidal fibroblasts in the CCl4-induced fibrosis of the rat liver.

Development and regression of liver fibrosis and cirrhosis induced by CCl4 in male F-344 rats were strictly followed during and after an 8-week treatment. The relative amount of collagen was measured by morphometry and the number of glycosaminoglycan (GAG) containing fat storing cells was counted at each time point. The expression of proteoglycan genes (decorin, versican and BPG-5 HSPG) was studied in parallel with the development of cirrhosis. Collagen content of the liver as well as the number of GAG-containing mesenchymal (fat storing) cells increased in parallel until two weeks after the cessation of CCl4 treatment. Later, both the collagen content and the number of GAG-containing cells decreased in parallel and significantly. Proteoglycan gene expression in the nonparenchymal fraction of liver cells indicated an active proteoglycan synthesis in the course of the development of cirrhosis. It is concluded that modified Ito (fat storing) cells synthesize proteoglycans and play an important role in the formation of connective tissue fibers in liver fibrosis.

The effect of D-penicillamine on CCl4-induced experimental liver cirrhosis.

The effect of D-penicillamine (Pe) on liver fibrosis-cirrhosis induced by chronic CCl4 and phenobarbital (Pb) administration in Fischer 344 male rats was studied. Morphometric analysis did not reveal a decrease in the amount of connective tissue fibers after Pe-treatment. Compared to the CCl4 and Pb-treated control groups, Pe had no significant effect on the concentrations of hydroxyproline, a parameter of collagen degradation, either; however, it increased the glycosaminoglycan concentrations. Lymphocyte stimulation by Con-A in the Pe-treated groups did not differ from that of the CCl4 and Pb-treated ones. According to our studies, Pe-treatment was ineffective in rats with liver fibrosis-cirrhosis induced by CCl4 and Pb administration. It seems that Pe can be effective only in the cirrhosis types accompanied by a considerable copper accumulation due to suppression of the toxic effects of copper.

Modulation of membrane phenotype, matrix adhesion and microinvasiveness of metastatic tumour cells by HUdR.

The effect of HUdR, proved to be anti-metastatic in vivo, was studied in vitro on cell proliferation, nucleoside uptake, membrane fluidity, expression of galactosylated glycans and proteoglycans in metastatic HM tumour cells. The observed increase in membrane fluidity and the suppression of nucleoside transport were early events of the HUdR action followed by decrease of galactosylated glycan and HSPG expression. However, these changes did not influence the proliferation capacity of the cells at the concentrations studied. As a consequence of the membrane alterations a reduced adhesiveness and spreading on extracellular matrix components was detected. In addition, the HUdR treated HM cells showed reduced capacity to invade fibroblast monolayers in vitro. Based on these observations, HUdR could be the prototype of new anti-metastatic agents acting at the level of tumour-host interaction.

Modification of the inhibitory effects of CCl4 on phospholipid and protein biosynthesis by prostacyclin.

CCl4 induced cellular injury and its modification by prostacyclin (PGI2) was studied in cultured rat hepatocytes. Biosynthesis of both intracellular and serum proteins and that of phospholipids decreased upon CCl4 treatments (IC50 7.0, 2.5 and 3.2 mM, respectively). After 1 hr exposure of the cells to CCl4, the reductions in the biosynthesis increased further with time. PGI2 treatments (10(-5)-10(-9) M) of the hepatocytes subsequent to CCl4 poisoning resulted in partial recovery from the cell injury evaluated at the fifth hour of the experiment. Optimal effects of PGI2 were found at a concentration of 10(-7)-10(-8) M, while higher and lower concentrations offered less protection. Upon the addition of CCl4 a higher catabolic rate of PIP2 and an increased formation of inositol phosphates were observed. This alteration was shown to precede the defects in the labelling of the major phospholipid components. Furthermore, these changes were circumvented in the presence of PGI2. Thus, PIP2 metabolism appears to be a critical process in the mechanism of this type of cellular injury and its protection by PGI2.

[The effect of D-penicillamine on experimental liver cirrhosis induced by CCl4]. / A D-penicillamin hatása a CCl4 kiváltotta kísérletes májcirrhosisra.

Authors examined the effect of D-penicillamin (Pw) on liver cirrhosis induced by chronic CCl4 and phenobarbital (Pb) treatment in Fischer 344 rats. Morphometric analysis of quantity of connective tissue fibres did not show decrease on the effect of Pe treatment. Quantity of hydroxiproline, which is one of the parameters of coll ahen decrease, did not change significantly on effect of drug, but only compared to CCl4 and Pb treated control. Quantity of glycosaminoglycan showed increase following Pe treatment. Lymphocyte stimulation by Con-A was different in CCl4 and Pb and Pe treated groups, respectively. According to our examinations in case of liver fibrosis cirrhosis induced by CCL4-PB treatment in rats the Pe treatment proved to be unsuccessful. It seems that Pe is effective only in forms of cirrhosis accompanied by significant copper accumulation, by decrease of toxic effects of copper.

Comparative study on Lewis lung tumour lines with 'low' and 'high' metastatic capacity. III. Glycosaminoglycan synthesis, transport and degradation in cell lines.

The glycosaminoglycans (GAGs) of low (LM) and highly metastatic (HM) cell lines of the Lewis lung tumour (3LL) were compared using [3H]glucosamine labelling techniques. The GAGs isolated from nuclei, cytoplasm, pericellular fractions and medium were analysed by cellulose acetate electrophoresis and by digestion with specific enzymes, and the following conclusions were drawn. 1. Increased cellular uptake and incorporation of [3H]glucosamine into glycoconjugates of the cytoplasm was a typical feature of the highly metastatic cell line after a 48-h labelling. However, there was no elevated radioactivity in glycolipids. 2. Radioactivity of the purified GAGs was two and three times higher in nuclear and cytoplasmic fractions of HM cells than in those of LM cells. There was much less difference between the two cell lines in the pericellular fractions. 3. A definite change from chondroitin sulphate to dermatan sulphate dominancy was recorded in each GAG fraction. Higher heparan sulphate labelling was observed in the cytoplasmic and pericellular GAGs of HM cultures. 4. In the post-labelling period about three times more GAG was present in the extracellular compartment of the HM cultures compared with the LM cultures. 5. In the LM cultures the total GAG-associated radioactivity decreased by 73 per cent in the 48-h chase period whereas in the HM cultures it decreased by only 30 per cent. This indicates a higher rate of GAG degradation in the LM cultures.

Glycosaminoglycan containing fat-storing cells in hepatic fibrogenesis.

Male F-344 rats were treated for 10 weeks either with CCl4 (0.2 ml/kg, per os, twice a week) or with CCl4 (same as above) and phenobarbital (0.2 g/l in drinking water). Liver fibrosis and cirrhosis developed in both treated groups, and was confirmed histologically. Cirrhosis was more frequent after the CCl4 + phenobarbital treatment. The collagen content of the liver, measured by morphometry and biochemically, was significantly higher in the animals of the group treated with CCl4 + phenobarbital than in the animals treated only with CCl4. Specially altered fat-storing cells (Ito cells) were found in the periportal and septal fibrotic areas in direct proportion to the amount of fibrosis and cirrhosis. They were identified as altered fat-storing cells by their desmin content and Vitamin A storing capability. This study demonstrated that these cells were enlarged and contained neutral fat, lipofuscin and PAS-positive material. The potential role of GAG-containing FSC in fibrogenesis is discussed.

Comparative study on Lewis lung tumor lines with 'low' and 'high' metastatic capacity. II. Cytochemical and biochemical evidence for differences in glycosaminoglycans.

The enhanced metastatic capacity of an in vivo selected Lewis lung tumor line (LLT-HH) was correlated with changes in cell-associated glycosaminoglycans (GAG) using ultrastructural cytochemistry, flow cytometry and biochemistry. The increase in highly sulphated GAG content on the cell membrane of LLT-HH cells compared to the parent LLT cells was demonstrated cytochemically. Using in vitro [3H]glucosamine labelling of GAG components it was shown that the LLT-HH cells were characterized by a high production of heparan sulphate while the parent LLT line had a high hyaluronic acid-chondroitin sulphate production. The high metastatic phenotype is accompanied by an altered production of cell-associated GAGs.

Morphological investigations on co-cultures of rat hepatocytes and the neonatal liver cell line RL 19.

Hepatocytes isolated from mature rats were cultivated together with proliferating liver cells derived from newborn rats (RL 19 line). The hepatocytes of the co-cultures were viable for long period and preserved their glucose-6-phosphatase activity and glycogen content longer than the mature rat hepatocytes cultured alone. Electron microscopy revealed that the RL 19 cells were covering the hepatocytes and growing beneath them. Both the RL 19 cells and the adult hepatocytes established tight contact with each other, but between the two cell types there was always a gap bridged by microvilli and resembling Disse's space. It is assumed that the envelope formed by the RL 19 cells may have a role in the maintenance of the differentiated hepatocyte structure.