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Two independent dedifferentiated variants, H5 and FaoflC2, derived from the Reuber H35 hepatoma, produce trans-acting diffusible substances(s) that extinguish the expression of liver-specific proteins when hybridized with a well-differentiated cell line of the same origin (Fao and Fu5-5, respectively). H5 x Fao hybrids show total and stable extinction of four liver functions and clonal variability in the expression of three others. FaoflC2 x Fu5-5 hybrids are initially flat (like FaoflC2 cells), and die in glucose-free medium where survival requires expression of hepatic gluconeogenic enzymes, but then evolve to hepatoma-like and finally round morphology; these latter cells express all liver functions analyzed including the gluconeogenic enzymes. Two exceptional clones that remained flat long enough for complete analysis showed extinction of all hepatic functions not expressed by FaoflC2 cells. We conclude that this transitory extinction reflects the action and then loss of extinguishing factor(s) contributed by FaoflC2. When crossed with BW1-J mouse hepatoma cells. FaoflC2 causes stable extinction of mouse aldolase B. We propose that production of extinguishing factor(s) is the rule for dedifferentiated variants.

A cross has been performed between dedifferentiated rat hepatoma cells and the differentiated cells from which they were derived. 10 hybrid clones, containing the complete chromosome sets of both parents, show extinction of 4 liver-specific enzymes: tyrosine aminotransferase (E.C. 2.6.1.5), alanine aminotransferase (E.C. 2.6.1.2), and the liver-specific isozymes of alcohol dehydrogenase (E.C. 1.1.1.1) and aldolase (E.C. 4.1.2.13). Moreover, the 4 hybrid clones examined do not produce albumin . The only function of the differentiated parent which is not extinguished in the hybrid cells is inducibility of the aminotransferases. For 3 of the hybrid clones, extinction of 3 of the 4 enzymes is incomplete, but these clones do not differ in modal chromosome number from those which show more complete extinction of the enzymes. Subcloning of several of the hybrids revealed that the phenotype of the hybrids is very stable; 4 subclones showing reexpression of intermediate levels of the enzymes are characterized. These results show that dedifferentiation of the parental cells is not due to the simple loss of some factor required for the maintenance of expression of differentiated functions, and suggest that dedifferentiation is due to the activation of some control mechanism, whose final effect is negative, and which may be a part of the epigenotype of the embryonic hepatocyte.

Non-linear dynamics, often chaotic, are now accepted as the normal way in which major physiological functions proceed, allowing adaptation. The length of telomeres, the chromosome endings, is critical in limiting cell lifespan and in controlling subsets of downstream genes. Telomere length dynamics in tumoral cells is the net result of mitotic erosion of telomeric DNA and telomere repair by the enzyme telomerase. We observed telomere length oscillations in the long-lived hepatoma Fao cell line, for forty three 6-day passages in culture. Telomerase activity oscillated with similar frequency and opposite amplitude. We mapped the combined data of telomere erosion and telomerase activity. There was reverse symmetry between consecutive periods of 10 passages. Thus, telomere length and telomerase activity can be considered to be a single complex system with strong reciprocal regulation.

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A 23-kilobase-pair segment of DNA containing the entire mouse serum albumin gene as well as 2.2 kilobase pairs of 5' and 4.3 kilobase pairs of 3' flanking sequences has been introduced into pSV2dhfr, a plasmid in which expression of the mouse dihydrofolate reductase cDNA is under the control of simian virus 40 sequences. This vector, pSV2dhfr-alb, was used to transfect differentiated and variant dedifferentiated rat hepatoma cells. Nine independent clones of transfected differentiated cells secrete considerable amounts of mouse albumin, while the expression of the normal rat albumin is the same as in nontransfected cells. In contrast, only small amounts of mouse and rat albumin are produced by transfected dedifferentiated cells. The amounts of albumin mRNA present in the cells are consistent with the amounts of albumin produced. These results show that a transfected gene can be regulated in a fashion consistent with the overall differentiation profile of the cell.