Ubiquitous, heritable damage in cell populations that survive treatment with methotrexate

A permanent line of mouse embryo fibroblasts was treated with concentrations of the anticancer drug methotrexate (MTX) that left 20–50% surviving colonies. The surviving population initially multiplied at a much slower rate than controls after subculture in the absence of the drug, and required 9–12 days of serial subculture, with selective growth of the faster growing cells, to approximate the control rate. To determine the distribution of growth rates of cells in the original posttreatment populations, many single cells were isolated in multiwell plates immediately after the treatment period, and the resulting clones were serially subcultured. Most of the control clones underwent about 2 population doublings per day (PD/D). Almost all the survivors of MTX treatment multiplied at heterogeneously reduced rates, ranging from 0.6 PD/D to as high as control rates for a very few clones. They maintained the reduced rates through many subcultivations. The heritability of the reduced growth rates indicates that most cells that retain proliferative capacity after treatment with MTX carry random genetic damage that is perpetuated through many divisions of their progeny. Similar results have been described for cells that survive x-irradiation, and suggest random genetic damage is a common occurrence among cells in rapidly growing tissues that survive cytotoxic treatment. It also occurs in serial subcultures of cells that had been held under the constraint of confluence for extended periods, which suggests that the accumulation of random genetic damage to somatic cells during aging of mammals underlies the reduction of growth rate and function of the cells that characterizes the aging process.

Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome

Following transcription and splicing, each mRNA of a mammalian cell passes into the cytoplasm where its fate is in the hands of a complex network of ribonucleoproteins (mRNPs). The success or failure of a gene to be expressed depends on the performance of this mRNP infrastructure. The entry, gating, processing, and transit of each mRNA through an mRNP network helps determine the composition of a cell's proteome. The machinery that regulates storage, turnover, and translational activation of mRNAs is not well understood, in part, because of the heterogeneous nature of mRNPs. Recently, subsets of cellular mRNAs clustered as members of mRNP complexes have been identified by using antibodies reactive with RNA-binding proteins, including ELAV/Hu, eIF-4E, and poly(A)-binding proteins. Cytoplasmic ELAV/Hu proteins are involved in the stability and translation of early response gene (ERG) transcripts and are expressed predominately in neurons. mRNAs recovered from ELAV/Hu mRNP complexes were found to have similar sequence elements, suggesting a common structural linkage among them. This approach opens the possibility of identifying transcripts physically clustered in vivo that may have similar fates or functions. Moreover, the proteins encoded by physically organized mRNAs may participate in the same biological process or structural outcome, not unlike operons and their polycistronic mRNAs do in prokaryotic organisms. Our goal is to understand the organization and flow of genetic information on an integrative systems level by analyzing the collective properties of proteins and mRNAs associated with mRNPs in vivo.

Phosducin is a ubiquitous G-protein regulator.

Phosducin is a 33-kDa cytosolic regulator of G-protein-mediated signaling that has previously been thought to be specific for retina and pineal gland. In this study, we show widespread tissue distribution of phosducin by the amplification of its cDNA and the detection of two different transcripts in Northern analyses in liver, lung, heart, brain, and retina. On the protein level, phosducin could be detected in 12 bovine tissues by immune precipitation and subsequent Western analysis using anti-phosducin antibodies generated in two different species. Masking of phosducin in direct Western blots appears to explain the failure to detect phosducin in earlier studies. The concentration of phosducin in bovine brain was calculated in the range of 10 pmol/mg total cytosolic protein (approximately 1 microM), whereas in the other tissues, it was slightly less. In these concentrations, phosducin inhibited receptor-stimulated adenylyl cyclase activity in cell membranes by about 50%. Taken together, our results indicate that phosducin is a ubiquitous regulator of G-protein function.

The histone fold: a ubiquitous architectural motif utilized in DNA compaction and protein dimerization.

The histones of all eukaryotes show only a low degree of primary structure homology, but our earlier crystallographic results defined a three-dimensional structural motif, the histone fold, common to all core histones. We now examine the specific architectural patterns within the fold and analyze the nature of the amino acid residues within its functional segments. The histone fold emerges as a fundamental protein dimerization motif while the differentiations of the tips of the histone dimers appear to provide the rules of core octamer assembly and the basis for nucleosome regulation. We present evidence for the occurrence of the fold from archaebacteria to mammals and propose the use of this structural motif to define a distinct family of proteins, the histone fold superfamily. It appears that evolution has conserved the conformation of the fold even through variations in primary structure and among proteins with various functional roles.

Conditional expression of the ubiquitous transcription factor MafK induces erythroleukemia cell differentiation.

Transcription factor NF-E2 activity is thought to be crucial for the transcriptional regulation of many erythroid-specific genes. The three small Maf family proteins (MafF, MafG, and MafK) that are closely related to the c-Maf protooncoprotein constitute half of the NF-E2 activity by forming heterodimers with the large tissue-restricted subunit of NF-E2 called p45. We have established and characterized murine erythroleukemia cells that conditionally overexpress MafK from a metallothionein promoter. The conditional expression of MafK caused accumulation of hemoglobin, an indication of terminal differentiation along the erythroid pathway. Concomitantly, DNA binding activities containing MafK were induced within the MafK-overexpressing cells. These results demonstrate that MafK can promote the erythroid differentiation program in erythroleukemia cells and suggest that the small Maf family proteins are key regulatory molecules for erythroid differentiation.