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The retention of the two parental mitochondrial DNAs has been investigated in a large number of mouse-human cell hybrids segregating either mouse or human chromosomes, by using a highly sensitive and specific method for detection of the DNA; the results have been correlated with the karyotype and isozyme marker pattern in the same hybrid lines. In e hybrids examined, a consistent pattern was observed for the type of mitochondrial DNA retained: the mitochondrial DNA of the parent whose chromosomes were segregated from the nucleus was undetectable or present in marginal amounts. This was true also of hybrids containing a complete set of the segregating chromosomes in the total or a large fraction of the cell population. Previous investigations using hybrid cells derived from fusion of cells from mice and humans (1-3), rats and humans (3), or mice and hamsters (4) have established in these hybrids a correlation, though not very strong, between chromosome and mitochondrial DNA (mtDNA) segregation. However, in none of these studies has a detailed karyological analysis of the hybrids been performed to establish the rules that govern the retention of the two parental mtDNAs in interspecific cell hybrids. Thus, it is still not known whether the disappearance of mtDNA of one parental species in the hybrid cell depends on the loss of one chromosome or set of chromosomes of that species or on an imbalance of chromosomes of the two species or on a more complex regulatory phenomenon.A large series of hybrids between the human cell line HT-1080 and mouse cells has been isolated (5). These hybrids tend to lose chromosomes of either species depending on the mouse parent, but many of them retain in a relatively stable form a large number of chromosomes of both species. In this paper we present the results of a parallel systematic investigation of the mtDNA composition, karyotype, and isozyme marker pattern in a fairly large number of these hybrids. By using a highly sensitive and specific method for detection of mtDNA of the two parental species, we show that, in all hybrids analyzed, the mtDNA of the species being segregated from the nucleus was undetectable or present only in minute amounts. METHODSCell Hybrids Used. The methods for production and selection of mouse-human hybrid cell lines that lose chromosomes of either species have been described (5-7). A complete list of all hybrids analyzed in this study and the parental cell lines of each are presented in Table 1. Hybrids in group 1 were formed between a thioguanine-resistant variant (6TG) of the human cell line HT-1080 (5) and either mouse peritoneal macrophages or cells derived directly from the solid mouse teratocarcinoma OTT-6050 and were selected in hypoxanthine/aminopterin/ thymidine (8). Hybrids in group 2 were formed between the cell line HT-1080 and the contact-inhibited continuous mouse cell line THO-2, which is a ouabain-resistant, hypoxanthine phosphoribosyltransferase-deficient 3T3 derivative (9), and were selected in hypoxanthine/aminopterin/thymi...

Line A of Walker 256 carcinoma implanted in the muscle adjacent to the tibia of young (6 weeks) and adult (9 months) male rats invaded the bone. Osteolysis and reactive growth were greater in the bone of young animals than in adults. Ethane-1-hydroxy-1,1-bisphosphonate prevented bone lysis and tumor invasion of the cortex both in young and adult animals. This model may be useful for studies of age-related differences in tumor infiltration into the bone and for investigating drug effects on this process.

The specific activity of mouse neuroblastoma choline-O-acetyltransferase (EC 2.3.1.6.) increased 5.7-fold when the rate of cell division was restricted (as compared to cells kept rapidly dividing for 9 days); the specific activity of mouse neuroblastoma thymidylate synthetase increased 2.4-fold when nondividing cells again entered the logarithmic phase of cell growth. The highest specific activities for choline-O-acetyltransferase and lowest specific activities for thymidylate synthetase were obtained from cultures where cell division was restricted; the opposite result was observed when the cells were growing rapidly. Thus, the regulation of these two enzymes is out of phase with respect to each other and is dependent on the rate of cell division. The inverse relationship for the regulation of these two enzymes is discussed in relation to the needs of mitotic versus differentiated neuroblastoma cells.Mouse neuroblastoma 0-1300 tumor cells in culture synthesize acetylcholine and norepinephrine and also generate spontaneous action potentials and action potentials in response to acetylcholine and electrical stimulation (14). Further, the tumor cells in culture possess a permease for dopamine uptake, and electron micrographs show clusters of dense-core vesicles in nerve terminals (3). Blume et al. (5) recently demonstrated the regulation of mouse neuroblastoma acetylcholinesterase specific activity in cell culture when the rate of cell division was restricted, and showed that the regulatory mechanism for acetylcholinesterase was inversely related to the rate of cell division. Highest acetylcholinesterase specific activity was obtained in cultures containing nondividing cells.We Clone N-18 of mouse neuroblastoma C-1300 was grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum, sodium penicillin G (25 units/ml), and streptomycin sulfate (25 Mug/ml) in Falcon flasks or Petri dishes at 370C in an atmosphere of 10% C02-90% air at 100% humidity.For the choline-O-acetyltransferase experiments, 250-ml Falcon flasks (75 cm2) were incubated with 2 X 106 cells, grown to confluency and maintained confluent for 3 weeks. The medium was changed in these flasks on alternate days for the first 2 weeks and then daily for the last 7 days. At day zero of the choline-O-acetyltransferase experiment, the medium in each of the flasks was aspirated, and the cells were dissociated from the plastic surface by incubation in Ca++-and Mg++-free saline D-1 medium, supplemented with 5.6 mM glucose and 59 mM sucrose (modified D-1) for 15 min. About half of the cells from each flask could be tapped off into this medium. The cells were then centrifuged, resuspended in modified Eagle's Medium, and counted with a hemocytometer by the nigrosin method to determine viability (6). Plastic Petri dishes, 100-mm size, containing 10 ml of modified

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