Spontaneous metastasis of rat liver epithelial cells transformed with v-raf and v-raf/v-myc: association with different phenotypic properties.

Cloned v-raf, v-raf/v-myc, and spontaneously transformed rat liver epithelial (RLE) cell lines were examined for meastatic capability in nude mice, using the LacZ gene as a marker for quantitation of micrometastases. Six cloned lines (R3611-T lines) derived from nude mouse xenografts of the v-raf transformed R3611-3 cells displayed variable metastatic capabilities. Three of six subcutaneously inoculated R3611-TlacZ lines produced spontaneous lung metastasis in nude mice. One of the lines, R3611-T2lacZ was highly efficient at metastatic conversion and produced more lung colonies than a faster growing v-raf/v-myc-transformed RJ2-14lacZ line. The spontaneously transformed RLElacZ line (C4T) was nonmetastatic, although it produced larger subcutaneous tumors than the metastatic R3611-T2lacZ line. Metastatic conversion correlated with upregulation of urokinase-type plasminogen activator receptor RNA expression and downregulation of plasminogen activator inhibitor-1, collagen alpha1 (I), and cytokeratin 14 (K14) RNA expression. These findings indicate that proteolytic activities associated with plasminogen activation play a role in the metastatic development in this model. Decreased production of extracellular proteins and cytoskeletal changes associated with lack of K14 expression are also likely to have contributed to the metastatic conversion of the RLE transformants.

Modulation of keratin 14 and alpha-fetoprotein expression during hepatic oval cell proliferation and liver regeneration.

Keratin 14 (K14) expression has recently been demonstrated in cell lines of non-parenchymal hepatic origin (Bisgaard et al., 1993, Mol. Carcinog., 7:60-66; Bisgaard et al., 1991, J. Cell. Physiol., 147:333-343). These cell lines are thought to represent a progeny of a dormant stem cell compartment present in the adult rat liver, which may participate in the restoration of the liver mass after experimental liver injury. Utilizing a combination of 2-acetylaminofluorene (2-AAF) administration and partial hepatectomy to activate liver regeneration by proliferation of oval cells, we examined the modulation of K14 as well as alpha-fetoprotein (AFP) expression in proliferating oval cells and lineages hypothesized to be derived herefrom. We showed by Northern blot and in situ hybridization analyses that K14 and AFP transcripts were initially accumulating in epithelial cells located in subsets of ductal structures in the portal areas. As oval cells infiltrated the liver parenchyma, K14 transcripts were detected in oval cells, in foci of small basophilic hepatocytes, and in structures resembling glandular intestinal-type epithelium. AFP was expressed in oval cells, and at low but detectable levels in foci of basophilic hepatocytes, but not in glandular intestinal-type epithelium. Neither K14 nor AFP transcripts were detected in bile ducts or mature hepatocytes at any time during oval cell proliferation and reconstitution of the liver mass. To further study the modulation of K14 and AFP expression we utilized an in vitro model in which spontaneous transformation of rat liver epithelial (RLE) cells appeared to mimic the process of early differentiation along the hepatic lineage in vivo. We demonstrated that undifferentiated RLE cells at a late passage expressed K14 and vimentin, whereas transformation and differentiation to hepatoblast-like progeny resulted in an abrogation of K14 and vimentin expression and an induction of K18 and AFP. We propose that K14 and AFP are sequentially modulated in subpopulations of oval cells involved in the ongoing reconstitution of the liver mass.

Phenotypic modulation of keratins, vimentin, and alpha-fetoprotein in cultured rat liver epithelial cells after chemical, oncogene, and spontaneous transformation.

Several lines of evidence have indicated that rat liver epithelial (RLE) cell lines may be related to a dormant stem cell compartment in the liver in vivo. We have demonstrated that keratin 14 (K14) is expressed together with vimentin in undifferentiated RLE cells. However, upon spontaneous transformation and differentiation to hepatoblast-like progeny the expression of these intermediate filaments (IF) is abrogated, while expression of another set of genes, among others keratin 18 (K18) and alpha-fetoprotein (AFP), is induced (Bisgaard et al., 1994, J. Cell. Physiol., in press). To better understand the mechanisms underlying IF expression during transformation and differentiation of RLE cells we examined the expression and regulation of IFs in clonal cell lines of chemically, oncogene, and spontaneously transformed RLE cells and their resulting tumors. These clonal lines provided a wide variety of tumor phenotypes including trabecular, solid and tubular adenocarcinomas, undifferentiated carcinomas, and spindle cell carcinomas. Northern blot analysis of the cell lines confirmed the differential expression of IF mRNAs. While keratin 8 (K8) was expressed at similar steady-state levels in all cell lines, K14 and vimentin but not K18 were expressed in the majority of cell lines chemically transformed with aflatoxin B1 or by transduction of oncogenes. In contrast, cell lines transformed spontaneously by prolonged passage in vitro expressed K18, while K14 and vimentin were absent. The keratin expression pattern in vitro was retained in the majority of the resulting tumors. However, the keratins expressed in vitro did not accurately predict the tumor phenotype in vivo. In particular, in tumors typed morphologically as adenocarcinomas, the keratin pair typically expressed in chemically transformed tumor cells was K8/K14, whereas K8/K18 was expressed in the tumors derived from spontaneously transformed cell lines. Finally we showed by nuclear run-on and in vitro translation analyses that the expression of K14, K18, and vimentin in transformed RLE cell lines was regulated at the transcriptional level, whereas that of K8 appeared to be posttranslational. These findings suggest that events controlling the differential expression of IF genes are involved in the processes leading to transformation and differentiation of the RLE cell lines. We conclude that the transformed RLE cell lines provide a valuable model to further examine the regulatory mechanisms involved in hepatic differentiation of undifferentiated "progenitor-like" RLE cells.