p53 cellular tumor antigen: analysis of mRNA levels in normal adult tissues, embryos, and tumors.

The relative levels of mRNA specific for the mouse p53 cellular tumor antigen were determined in various normal adult tissues, embryos, and tumors. All tumors studied contained concentrations of p53 mRNA well above those present in most normal tissues. Normal spleen, however, had p53 mRNA levels comparable to those found in some tumors, despite the fact that they contained barely detectable p53 protein. This apparent discrepancy was found to be due to the extremely rapid turnover rate of p53 in the spleen (half-life, approximately equal to 6 min). In developing fetuses, a marked reduction of p53 mRNA levels was manifest from day 11 onwards, whereas the levels during organogenesis (days 9 to 11) were comparable to those found in undifferentiated embryonic stem cells and in some tumors.     

The stereochemical course of phospho transfer catalyzed by adenylosuccinate synthetase. A reaction pathway via a phosphorylated intermediate with net inversion

The stereochemical course of phospho transfer in the reaction catalyzed by adenylosuccinate synthetase from rat muscle has been determined with chiral [gamma-17O,18O]GTP gamma S as a substrate. The stereochemical configuration of the product, inorganic thiophosphate, was determined by 31P NMR after the compound was stereospecifically incorporated into ATP beta S. The reaction goes with net inversion of configuration, which is the course for a single phospho transfer, even though 6-phospho-IMP is probably an intermediate on the normal reaction pathway (Liebermann, I. (1956) J. Biol. Chem. 223, 327-339). The breakdown of this intermediate goes by C-O bond cleavage and so is not a true phospho transfer step. Thus, inversion of configuration during the course of this ligase reaction is consistent with a single phospho transfer step in the overall reaction, the formation of the phosphorylated intermediate.     

F-actin aggregates in transformed cells contain alpha-actinin and fimbrin but apparently lack tropomyosin

Transformation-specific F-actin structures are examined in tumor cells after in vitro tumor cell growth alone or on an untransformed cell monolayer. In transformed cells F-actin aggregates near the ventral plasma membrane in close substrate adhesion areas contain the cytoskeletal proteins alpha-actinin and fimbrin but, unlike microfilament bundles, are not labeled with antibody against tropomyosin. By electron microscopy the dense ventral aggregates in transformed cells resemble stress fiber termini found at the membrane in normal cells. These transformed-cell cytoskeletal structures are not limited solely to substrate adhesion areas; they are also expressed at cell-cell contacts about 48 h after transformed cells are plated on untransformed cells. These specialized F-actin aggregates appear to be implicated in the processes of penetration of these transformed cells between adjoining untransformed cells in vitro.     

A model of proliferating cell populations with inherited cycle length

A mathematical model of cell population growth introduced by J. L. Lebowitz and S. I. Rubinow is analyzed. Individual cells are distinguished by age and cell cycle length. The cell cycle length is viewed as an inherited property determined at birth. The density of the population satisfies a first order linear partial differential equation with initial and boundary conditions. The boundary condition models the process of cell division of mother cells and the inheritance of cycle length by daughter cells. The mathematical analysis of the model employs the theory of operator semigroups and the spectral theory of linear operators. It is proved that the solutions exhibit the property of asynchronous exponential growth.     

Metastatic phenotype of human prostate tumor cells in athymic nude mice: Alteration by exposure to ethyl methanesulfonate and “reversion” by 5-azacytidine

Summary The human prostate tumor subline 1-LN-PC-3-1A (1-LN) is reproducibly metastatic in adult athymic nude mice. Cells surviving a brief in vitro exposure to ethyl methanesulfonate (EMS) exhibited a profound decrease in capacity for experimental lung metastasis in nude mice. Thirty days after EMS treatment, 1×10⁶ uncloned EMS-treated 1-LN cells (1-LN-EMS-10) were injected IV into groups of 6 to 8-week-old male athymic nude mice (BALB/cAnBOM). A median of 8.5 colonies/lung was observed among 20 1-LN-EMS-10-injected mice, which was significantly different from the median of 51 colonies/lung produced among 14 1-LN-injected mice (P=0.0002). This altered phenotype remained stable during 150 days of continuous culture. However, the 1-LN-EMS-10 cells were tumorigenic in 10/10 nude mice injected SC. Single lung tumor colonies recovered from 1-LN-EMS-10-injected mice and reinjected IV into nude mice produced medians of 32–63 colonies/lung. The altered metastatic phenotype resulting from treatment of 1-LN with EMS was reversed by exposure to a noncytotoxic dose of 5-azacytidine, but unaffected by a second exposure to EMS. Collectively these data demonstrate that the metastatic phenotype of these human tumor cells in athymic nude mice can be heritably altered by in vitro exposure to EMS and 5-azacytidine. Analysis of the mechanisms underlying these phenotypic changes may provide insight into parts of the complex process of tumor cell evolution.     

Proteoglycan extraction of sized cartilage particles

The relationship between cartilage thickness and proteoglycan extractability was examined. Bovine nasal cartilage slices (20, 100, and 500 micron thicknesses) were extracted with low-ionic-strength buffer and 4 M guanidine hydrochloride. The extractability of proteoglycans with both solutions depended on slice thickness. Thinner slices yielded greater amounts of proteoglycans. Sixty-three percent of the total cartilage uronic acid was extracted from 20-micron cartilage slices with low-ionic-strength buffer while only 7% was extracted for 500-micron slices. Each fivefold increase in cartilage surface area led to a threefold increase in uronic acid extraction with low-ionic-strength buffer. Extraction of proteoglycan aggregates was directly proportional to the cartilage surface area whereas extraction of non-aggregated proteoglycans, per surface area, increased with increasing cartilage thickness. These data are consistent with the hypothesis that proteoglycan aggregates are extracted mainly from the cartilage surface while non-aggregated proteoglycans diffuse from deep within the cartilage. Extraction with low-ionic-strength buffer occurred in two phases. There was an initial rapid loss of proteoglycans in which 1/3 to 1/2 of all proteoglycans eluting over 6 days were extracted during the first 30 min. Subsequent extraction was much slower with decreasing amounts extracted on each consecutive day. The initial rapid loss of proteoglycans was probably due to the steep osmotic-pressure gradient existing when the cartilage was placed in the low-ionic-strength buffer.     

Cell Surface Sialoglycoproteins of Cultured Rat Cerebellar Interneurons

Abstract: The sialoglycoproteins of cultures of relatively pure rat cerebellar interneurons were labelled by NaIO₄ oxidation/NaB ³H₄ reduction. The labelled molecules were analysed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate followed by fluorography. Faint labelling could be detected in three components if cells were labelled without any oxidation. In young cultures, oxidation by galactose oxidase alone failed to reveal any additional bands. After oxidation by NaIO₄ or galactose oxidase in the presence of neuraminidase, many more components were labelled. After NaIO₄ oxidation, about 80% of the cell-associated radioactivity could be removed by treating the cells with neuraminidase, which left the cells more than 95% viable. The majority of the bands seen after neuraminidase treatment were substantially reduced when compared with untreated controls, supporting a surface localisation of these molecules. Reproducible developmental changes were seen in the profiles of bands labelled by NaIO₄/NaB ³H₄ in time course studies of cultures up to 8 days in vitro . Some bands became more prominent, and others disappeared. The gel profiles of the neuron cultures were quite distinct from those of cerebellar astrocyte cultures, which contain all the cell types likely to be contaminants of the neuron cultures.     

Appearance of functional EGF receptor kinase during rodent embryogenesis.

Mouse and rat embryonic tissues at various stages of development were examined for epidermal growth factor (EGF) receptor kinase activity. The phosphorylated EGF receptor from embryonic tissues appeared as a band of mol. wt. 170 000 daltons on SDS gels. It was clearly demonstrable in the developing mouse fetus from 10 days of gestation onwards. The distribution of the EGF receptor kinase was studied in various tissues of 13 day mouse fetuses. The activity was apparent in the skin, developing skeletal muscles and various internal organs but was notably absent in the liver and brain. The amnion was found to be one of the richest sources of activity while the yolk sac was negative, and the placenta was weakly positive. In 16 day rat fetuses the distribution was quite similar to that of the 13 day mouse fetus. The liver acquired EGF receptor kinase activity by 18 days of gestation and had high activity in neonates. Phosphoamino acid analysis revealed that phosphotyrosine was the major labelled amino acid residue in the embryonic tissues. Thus, the EGF receptor of fetal tissues as studied by immune precipitation and phosphorylation appears to be a similar entity to that found in adult mammalian tissues. This functional EGF receptor kinase activity could first be detected at the time of onset of organogenesis.