Growth-associated gene expression is not constant in cells traversing G-1 after exiting mitosis

Analysis of gene expression following stimulation of growth-arrested cells has been the main approach for identification of growth-associated genes. Since the activation of these gene sequences is dependent on both the stimulatory agent and the state of quiescence of the cell, the activation and role of the same genes may be entirely different in non-growth arrested, actively proliferating cells. We have addressed the question of growth-associated gene expression during active growth by analyzing gene expression during G-1 of cells which have just exited mitosis without first leaving the cell cycle. We were able to isolate, by a non-inductive, drug free system, a population of highly synchronized Swiss 3T3 cells within mitosis (greater than 90%) in numbers sufficient to determine the pattern of expression of a large number of representative growth-associated genes. Our results show that after replating the mitotic cells into conditioned medium: (1) growth-associated gene expression is not constant during G-1 of actively proliferating cells, and (2) while a number of genes (e.g., JE, c-myc, ODC, p53, and histone) exhibited patterns of expression similar to that reported in the quiescent systems, others (e.g., nur-77, vimentin, calcyclin) exhibited patterns which were completely different. From these results, we can begin to construct a temporal map of G-1 progression during active growth.     

Partial reconstruction of the microvillus core bundle: characterization of villin as a Ca(++)-dependent, actin-bundling/depolymerizing protein

The brush border, isolated from chicken intestine epithelial cells, contains the 95,000 relative molecular mass (M(r)) polypeptide, villin. This report describes the purification and characterization of villin as a Ca(++)-dependent, actin bundling/depolymerizing protein. Then 100,000 g supernatant from a Ca(++) extract of isolated brush borders is composed of three polypeptides of 95,000 (villin), 68,000 (fimbrin), and 42,000 M(r) (actin). Villin, following purification from this extract by differential ammonium sulfate precipitation and ion-exchange chromatography, was mixed with skeletal muscle F-actin. Electron microscopy of negatively stained preparations of these villin-actin mixtures showed that filament bundles were present. This viscosity, sedimentability, and ultrastructural morphology of filament bundles are dependent on the villin:actin molar ratio, the pH, and the free Ca(++) concentration in solution. At low free Ca(++) (less than 10(-6) M), the amount of protein in bundles, when measured by sedimentation, increased as the villin: actin molar ratio increased and reached a plateau at approximately a 4:10 ratio. This behavior correlates with the conversion of single actin filaments into filament bundles as detected in the electron microscope. At high free Ca(++) (more than 10(-6) M), there was a decrease in the apparent viscosity in the villin-actin mixtures to a level measured for the buffer. Furthermore, these Ca(++) effects were correlated with the loss of protein sedimented, the disappearance of filament bundles, and the appearance of short fragments of filaments. Bundle formation is also pH-sensitive, being favored at mildly acidic pH. A decrease in the pH from 7.6 to 6.6 results in an increase in sedimentable protein and also a transformation of loosly associated actin filaments into compact actin bundles. These results are consistent with the suggestions that villin is a bundling protein in the microvillus and is responsible for the Ca(++)-sensitive disassembly of the microvillar cytoskeleton. Thus villin may function in the cytoplasm as a major cytoskeletal element regulating microvillar shape.     

Reevaluation of brush border motility: calcium induces core filament solation and microvillar vesiculation

The report that microvillar cores of isolated, demembranated brush borders retract into the terminal web in the presence of Ca(++) and ATP has been widely cited as an example of Ca(++)-regulated nonmuscle cell motility. Because of recent findings that microvillar core actin filaments are cross-linked by villin which, in the presence of micromolar Ca(++), fragments actin filaments, we used the techniques of video enhanced differential interference contrast, immunofluorescence, and phase contrast microscopy and thin-section electron microscopy (EM) to reexamine the question of contraction of isolated intestinal cell brush borders. Analysis of video enhanced light microscopic images of Triton- demembranated brush borders treated with a buffered Ca(++) solution shows the cores disintegrating with the terminal web remaining intact; membranated brush borders show the microvilli to vesiculate with Ca(++). Using Ca(++)/EGTA buffers, it is found that micromolar free Ca(++) causes core filament dissolution in membranated or demembranated brush borders, Ca(++) causes microvillar core solation followed by complete vesiculation of the microvillar membrane. The lengths of microvilli cores and rootlets were measured in thin sections of membranated and demembranated controls, in Ca(++)-, Ca(++) + ATP-, and in ATP-treated brush borders. Results of these measurements show that Ca(++) alone causes the complete solation of the microvillar cores, yet the rootlets in the terminal web region remain of normal length. These results show that microvilli do not retract into the terminal web in response to Ca(++) and ATP but rather that the microvillar cores disintegrate. NBD-phallicidin localization of actin and fluorescent antibodies to myosin reveal a circumferential band of actin and myosin in mildly permeabilized cells in the region of the junctional complex. The presence of these contractile proteins in this region, where other studies have shown a circumferential band of thin filaments, is consistent with the hypothesis that brush borders may be motile through the circumferential constriction of this “contractile ring,” and is also consistent with the observations that ATP-treated brush borders become cup shaped as if there had been a circumferential constriction.     

Characterization and biological activity of cloned simian virus 40 DNA fragments

The biological activity of fragments of the SV40 genome was determined by manual microinjection of the fragments into the nuclei of mammalian cells. Fragments of the SV40 A gene (that codes for the T antigens) were obtained either directly by digestion with restriction endonucleases or after cloning into plasmid pBR322. Three different biological activities were studied: expression of T antigen, induction of cell DNA synthesis, and, in a few cases, reactivation of repressed ribosomal RNA genes. By using a number of fragments with deletions in the various portions of the SV40 A gene, we have been able to conclude that: 1) the sequences from 0.65 to 0.51 map units are not needed for the induction of cell DNA synthesis; 2) the sequences from 0.42 to 0.17 map units are not needed for the induction of cell DNA synthesis; and 3) the critical sequences for the induction of cell DNA synthesis, 0.51 to 0.42 map units, are different from those necessary for the reactivation of repressed ribosomal RNA genes (0.39-0.33 map units). These results indicate that the information for these two fundamental processes of cell proliferation resides in two separate and distinct domains of the SV40 A gene.     

Hypersensitivity and reduced inhibition of DNA synthesis in ataxia telangiectasia lymphoblasts treated with low levels of neocarzinostatin

The effects of neocarzinostatin (NCS) on lymphoblastoid cell lines (LCLs) established from ataxia telangiectasia (A-T) were determined. A-T lymphoblasts were found to be hypersensitive to low levels of NCS as measured by cell growth and cell survival. On the other hand, A-T lymphoblasts failed to postpone DNA synthesis to the same degree as normal lymphoblasts following treatment with NCS. LCLs established from Nijmegen breakage syndrome (NBS) could be distinguished from ataxia and normal cell lines by their intermediate level of survival following exposure to NCS.     

Retinol-binding protein mRNA is induced by estrogen in the kidney but not in the liver

Vitamin A is mobilized from the liver and transported in plasma as retinol bound to retinol-binding protein (RBP). In addition to the liver, several extrahepatic tissues including the kidney have been shown to contain RBP mRNA. A study was conducted to explore the role of sex hormones in the regulation of RBP mRNA levels in the kidney compared to those in the liver. Treatment of female rats with a single dose of testosterone or chronic treatment with testosterone had only a slight effect on the steady-state level of RBP mRNA in the kidney and the liver. However, treatment of male rats with estrogen caused an increase in the steady-state level of RBP mRNA in the kidney but not in the liver. A single injection of 17 beta-estradiol, either 1.0 or 0.1 micrograms/g body weight, resulted in a rapid rise in the level of RBP mRNA in the kidney which was maximal at 3-6 h (fivefold induction) after treatment. In addition, treatment of ovariectomized female rats with estrogen also resulted in a rapid rise in the accumulated level of RBP mRNA in the kidney while having no influence in the liver. Finally, studies using the anti-estrogen drug, hydroxytamoxifen, resulted in blockage of the estrogen-related induction of RBP mRNA in the kidney, suggesting that the induction of RBP mRNA in the kidney by estrogen may be mediated by the nuclear estrogen receptor. Taken together these data suggest that the regulation of RBP mRNA, levels in the liver and kidney, at least with respect to estrogen, is different.     

Deletions in the SV40 late polyadenylation region downstream of the AATAAA mediate similar effects on expression in various mammalian cell lines.

A series of deletions in the SV40 late polyadenylation region was assayed by transient expression in a hamster fibroblast cell line. Because of differences in expression data between our results and the published results of another laboratory using a similar set of deletions introduced into a monkey kidney cell line, we studied our deletions in cells of different tissue-types and species (1). Deletion of the SV40 late polyadenylation region to 49 nucleotides downstream of the hexanucleotide AATAAA showed a small effect on gene expression, while further truncation of the region to 6 nucleotides downstream of the AATAAA showed an 85% drop in marker enzyme activity, protein levels and steady-state message levels. Another deletion in the same region, from base pair 10 to 15 past the AATAAA, which removes the wild-type site of RNA cleavage, showed a 50% drop in marker gene expression. The effects of these mutants on gene expression were similar in all of the cell lines tested and agree with other studies that DNA downstream of the AATAAA plays a role in efficient gene expression.