Changes in rat adrenal cyclic nucleotides during normal and neoplastic growth

In an attempt to correlate changes in cyclic nucleotide levels with in vivo growth of the rat adrenal gland we have measured adrenal cyclic AMP and cyclic GMP in normal, hyperplastic, and neoplastic rat adrenals. The first group of animals were subject to either unilateral adrenalectomy (ADX) or acute hypophysectomy 1 h prior to unilateral adrenalectomy (HADX). Cyclic nucleotides were measured in the contralateral adrenal post-operatively. In HADX rats cyclic GMP rose steadily throughout the 7 day study period, while ADX rats exhibited significant decreases in adrenal cyclic GMP. Cyclic AMP remained approximately 1.5 pm/mg tissue in HADX rats, while in ADX rats there was significant elevation of adrenal cyclic AMP at all time points. Cyclic GMP/cyclic AMP ratios remained constant in HADX animals; however, the growing adrenals of ADX animals exhibited depressed cyclic GMP/cyclic AMP ratios at all time periods.Adrenal hyperplasia was induced in a seond group of animals by a transplantable, corticotropin-secreting, pituitary tumor. Adrenals from age-matched animals served as controls. Adrenal cyclic AMP was significantly elevated in tumor-bearers at a time correspinding to the peak accumulation of adrenal weight, protein and DNA in these animals. In contrast, adrenal cyclic GMP in both tumor-beares and control animals fell steadily throughout the study period. Cyclic GMP/cyclic AMP ratios of control animals decreased from 2 to 3 weeks post-transplant remaining at the 3 week value during the period corresponding to rapid adrenal growth in tumor-bearers. The cyclic GMP/cyclic AMP ratio in the hyperplastic adrenals of tumor-bearers decreased steadily throughout their rapid growth period, suggesting a positive correlation between adrenal growth and depression of the cyclic GMP/cyclic AMP ratio.Cyclic nucleotide levels in neoplastic adrenals of rats bearing the transplantable adrenocortical carcinoma 494 were compared with cyclic nucleotides in normal rat adrenal glands. Cyclic AMP was not different in the two groups. However, the cyclic GMP content of neoplastic adrenals was significantly lower than that of normal adrenal tissue, causing a suppression of the cyclic GMP/cyclic AMP ratio in the neoplastic tissue. Thus, measurement of adrenal cyclic nucleotides in both hyperplastic and neoplastic rat adrenal glands suggests that adrenal growth in vivo may be characterized by a depression of the cyclic GMP/cyclic AMP ratio.     

Cyclin gene expression and growth control in normal and neoplastic human breast epithelium

Recent advances in defining the molecular mechanisms of cell cycle control in eukaryotes provide a basis for beter understanding the hormonal control of cell proliferation in normal and neoplastic breast epithelium. It is now clear that a number of critical steps in cell cycle progression are controlled by families of serine/threonine kinases, the cdks. These kinases are activated by interactions with various cyclin gene products which form the regulatory subunits of the kinase complexes. Several families of cyclins control cell cycle progression in G₁ phase, cyclins C, D and E, or in S, G₂ and mitosis, cyclins A and B. Recent studies have defined the expression and regulation of cyclin genes in normal breast epithelial cells and in breast cancer cell lines. Following growth arrest of T-47D breast cancer cells by serum deprivation restimulation with insulin results in sequential induction of cyclin genes. Cyclin D1 mRNA increases within 1 h of mitogenic stimulation and is followed by increased expression of cyclins D3 and E in G₁ phase, cyclin A in late G₁/early S phase and cyclin B1 in G₂. Similar results were observed following epidermal growth factor stimulation of normal breast epithelial cells. Other hormones—oestrogens and progestins—and growth factors—insulin-like investigated for their effects on G₁ cyclin gene expression. In all cases there was an excellent correlation between the induction of cyclin D1 mRNA and subsequent entry into S phase. Furthermore, growth inhibition by antioestrogens and concurrent G₁ arrest were preceded by an acute decrease in cyclin D1 gene expression. These observations suggest a likely role for cyclin D1 in mediating many of the known hormonal effects on cell proliferation in breast epithelial cells.     

Multiple forms of uridine kinase in normal and neoplastic rat liver

Two species of uridine kinase with molecular weights of approximately 120000 (I) and 30000 (II) have been identified in the rat liver system. Species I predominates in the 7-day postnatal and adult rat liver and increases in the regenerating remnant of the latter after partial hepatectomy; the concentration of species II is low in these tissues. Species I also predominates in the slow-growing hepatomas 5123D and 7800. In contrast, II is the predominant form in the foetal rat liver and accounts for 40% of the total activity in the rapidly growing Novikoff ascites hepatoma. In contrast to species II, which was stable, species I was inactivated by preincubation for 30min at 37 degrees C, before assay at 23 degrees C.     

Organization and expression of the chicken N-myc gene.

We cloned the chicken N-myc gene and analyzed its structure and expression. We found that it consisted of three exons with coding regions in exons 2 and 3. Comparison to mammalian N-myc genomic sequence indicated that nucleotide sequences of the 5'-flanking region, noncoding exon 1, and introns were not conserved, but coding and 3' noncoding sequences showed significant homology to mammalian N-myc. Alignment of deduced amino acid sequences of chicken and mammalian N-myc proteins revealed nine conserved domains interrupted by different lengths of nonhomologous sequences. Two of the domains were specific to N-myc proteins, and the other seven were common to c-myc proteins. Northern blot (immunoblot) and in situ hybridization analyses of 3.5-day-old chicken embryos revealed that high-level expression of the N-myc gene was confirmed to certain tissues, e.g., the central nervous system, neural crest derivatives, and mesenchyme of limb buds. In the beak and limb primordia, N-myc expression in the mesenchyme was higher toward the distal end, suggesting possible involvement in positional assignment of the tissue within the rudimentary structures.     

Altered cytokeratin expression and differentiation induction during neoplastic transformation of cultured rat liver cells by nickel subsulfide

Rat liver T51 B cells were maintained in the presence of low concentrations of Ni(H) derived from Ni₃S₂ for 3–I5 months in culture in order to monitor cytokeratin, differentiation, and transformation patterns. Nickel exposures caused irreversible, heritable juxtanuclear aggregates of cytokeratin CKSS, which increased in size and complexity with prolonged nickel exposure, eventually resembling Mallory bodies and expressing glutamyltransferase. Altered cytokeratin expression was accompanied by induction of differentiation, with markers of both bile ductular cells and hepatocytes, such as induction of cytokeratin polypeptides CK39 and CK49, cell morphology, and cytokeratin filament network changes, whereas control cultures similarly maintained for long periods in culture remained unchanged. Altered cytokeratin expression was also accompanied by acquisition of transformation markers—loss of density dependence, progression toward calcium independence, and (benign) growth in nude mice. Observed cytokeratin aberrations may be a factor in nickel carcinogenesis, in view of the known affinity of the metal for cellular structural proteins, especially keratin, which play a role in maintenance of cell behavior.Abbreviations CK cytokeratin - GGT gamma-glutamyltransferase - HCM standard calcium medium - LCM low calcium medium (Ca²⁺ = 0.02 mM) - MB mallory body - NI neoplastic index (ratio of growth of cells in LCM/HCM) - Ni ni(II) used as leachate from Ni₃S₂ - PBS phosphate-buffered saline - SB sodium butyrate     

Stability analysis of normal and neoplastic growth

A model for normal and abnormal tissue growth is defined by a system of differential equations. The model consists of a proliferating compartment, which is characterized by a decreasing growth rate, and a nonproliferating one. The compartments are also parametrized by their death and transfer rates. A stability analysis of the system shows in a precise way how abnormal growth may result from a perturbation of the parameters leading to a change in an equilibrium of the tissue size.     

Mutually exclusive expression of a helix-loop-helix gene and N-myc in human neuroblastomas and in normal development.

We have isolated a novel human gene encoding a helix-loop-helix (HLH) protein by molecularly cloning chromosome 1p36-specific CpG islands. The gene termed heir-1 was localized to the neuroblastoma consensus deletion at 1p36.2-p36.12. Its predicted protein is 95.8% identical to the mouse HLH462 protein and has clear homology to the mouse Id and Drosophila emc proteins. Heir-1 does not encode a basic DNA binding domain as found in basic HLH proteins. The gene is expressed specifically at high abundance in adult lung, kidney and adrenal medulla, but not in adult brain. Despite prominent heir-1 expression in adrenal medulla, which is a prime target for neuroblastomas, 10 out of 12 neuroblastoma-derived cell lines revealed very low levels of heir-1 mRNA. Low heir-1 expression was generally found in tumor cell lines with N-myc overexpression, whereas the two cell lines displaying high heir-1 levels did not overexpress N-myc. Mutually exclusive expression of both genes was also found by in situ hybridization in developing mouse tissues, particularly in the forebrain neuroectoderm. We conclude that heir-1 expression is reduced specifically in the majority of neuroblastomas and suggest an inverse correlation between heir-1 and N-myc expression in neuroblastoma tumors and in embryonic development.     

Increased expression of O-acetyl disialoganglioside synthase during rat liver fibrogenesis relates to stellate cell activation

The activation of the hepatic stellate cell (HSC) is a key step in liver fibrogenesis. Utilizing large scale sequencing of a 3'-directed cDNA library, we investigated expression profiles of quiescent and activated rat HSCs. During the activation process, O-acetyl disialoganglioside synthase (OAcGD3S) was identified as one of the significant upregulated factors. Upregulation of OAcGD3S in cultured HSCs was confirmed by both Northern and Western blot analyses. OAcGD3S expression in models of experimental liver fibrosis was investigated at the mRNA level using RT-PCR. The expression of OAcGD3S protein in activated rat HSCs and in experimental fibrotic livers was demonstrated by immunohistochemistry. In situ hybridization revealed OAcGD3S mRNA expression in areas of ductular proliferation. Furthermore, O-acetyl GD3 protein was detected in activated rat HSCs and human cirrhosis livers. This study shows that OAcGD3S is strongly expressed during liver fibrogenesis and HSCs seem to be the major cellular sources of OAcGD3S in the liver.     

Protein phosphorylation of the smooth and rough endoplasmic reticulum in normal and neoplastic liver of the rat.

Smooth and rough endoplasmic reticulum from rat liver and hepatomas exhibited endogenous protein kinase activity independent of adenosine 3':5'-monophosphate. The phosphorylation of smooth membranes by this process was consistently higher than that of rough membranes. When histone was added along with the smooth endoplasmic reticulum, cyclic AMP stimulated protein phosphorylation. Analysis of membrane-phosphorylated proteins by gel electrophoresis showed 5 major phosphorylated bands with estimated molecular weights of 155 000, 62 000, 50 000, 46 000 and 43 000, whereas major bands having estimated molecular weights of 62 000, 50 000 and 43 000 were found in membranes of the smooth endoplasmic reticulum of the Morris hepatoma 5123 C. Since previous studies in this and other laboratories have demonstrated the similarity of the protein components of membranes of the endoplasmic reticulum of normal liver and hepatoma, our findings indicate an inability of the protein kinase of hepatoma intracellular membranes to phosphorylate protein species that are found in membranes of both liver and the neoplasm.     

Increased expression of mature cathepsin B in aging rat liver

Senescence has been proposed as an important safeguard against neoplasia. One of the hallmarks of cellular senescence in vitro as well as human aging in vivo is a reduced intracellular protein catabolism. The pathways affected and the mechanisms responsible for the decrease in overall protein turnover in aging cells are not well understood. Our aim was to determine whether or not expression of one of the major hepatic lysosomal cysteine peptidases, cathepsin B, changes during aging of Sprague-Dawley rats. Cathepsin B activity was assessed in whole rat liver homogenates, and was found to be increased fourfold (P< or =0.001) in aged livers compared with younger counterparts. This was paralleled by an at least a twofold increase in mature cathepsin B protein. Nonetheless, Northern blot analysis of total liver RNA revealed no change in steady-state levels of cathepsin B mRNAs. These findings seem to contradict the present dogma according to which aging tissues have a reduced intracellular capacity to catabolise proteins. We propose that our earlier observation of the accumulation of T-kininogen, a potent but reversible cysteine peptidase inhibitor, in aging rat liver may provide a plausible explanation for this discrepancy.     

Regulation of protein disulfide isomerase gene expression in brain and liver during rat development

A 4-fold increase in protein disulfide isomerase (PDI) mRNA is observed in brain of 10 days-old rats and in liver of 20 days-old foetuses when compared with 20 days-old (brain) and 18 days-old (liver) foetuses respectively. During further postnatal development, the mRNA for PDI decreases in both organs to the initial values present in foetuses and remains practically unchanged in brain till the adult. By contrast in liver by 35-40 days after birth, and coincident with sexual maturation, there is a 2.5-fold increase in PDI mRNA that is maintained by 55 days (adult). These results clearly show that protein disulfide isomerase gene expression is differentially regulated in liver and brain during rat development.     

Polyamine acetylations in normal and neoplastic growth processes

Summary The expression patterns of cytosolic and nuclear polyamine acetyltransferases were studied in normal and neoplastic growth processesin vivo andin vitro to evidentiate the roles played by these enzymes in cell proliferation. In regenerating liver, cytosolic spermidine/spermine N¹-acetyltransferase showed similar augments of mRNA level and enzymatic activity during the prereplicative period (4–8 h), whereas spermidine N⁸-acetyltransferase activity increased later (24 h) when DNA synthesis was maximally enhanced. In fibroblasts continuously dividing, the messenger for spermidine/spermine N¹-acetyltransferase rapidly accumulated after serum-stimulation. In cultured Morris hepatoma cells stimulated to logarithmic growth, spermidine N⁸-acetyltransferase activity remained at plateau for 1 day declining thereafter, while spermidine/spermine N¹-acetyltransferase activity immediately decreased. In Yoshida AH-130 hepatoma cells transplanted in rat peritoneum, spermidine N⁸-acetyltransferase and spermidine/spermine N¹-acetyltransferase activities rose, respectively, in concomitance with elevated proliferation-rate and quasi-stationary phase of growth. Since the expression of cytosolic and nuclear acetyltransferases underwent different temporal activation, an involvement of these enzymes in separate metabolic processes controlling normal and neoplastic growth may be suggested.