Harold L. Moses

Effect of cell proliferation on levels and diversity of poly(A)-containing mRNA.

The relationship between cell proliferation and the amount and diversity of polyribosome-associated poly(A)-containing messenger RNA [poly(A)+mRNA]has been investigated using a cloned AKR-mouse embryo cell culture system. The following results were obtained. First, an early response to the stimulation of proliferation of AKR-2B cells in culture is a rapid increase in the rate of accumulation of polyribosomal poly(A)+ mRNA. This results in a large increase in the total poly(A)+ mRNA content of rapidly proliferating cells compared to that found in resting cells. Second, the total amount of unique DNA sequence contributing to the poly(A)+ mRNA populations of both growing and resting cells is not detectably different. This corresponds to 9000-11,000 diverse gene equivalents of DNA and represents the transcription of 0.8-0.9% of the haploid mouse genome. Third, most of the increased poly(A)+ mRNA content of growing cells (greater than 90%) reflects an increased rate of production of polysomal mRNA species which are also found in resting cells. Fourth, growing cells appear to contain some species of poly(A)+ mRNA which are either absent or present in very low concentrations in non-growing cells. Within the limits of detection, however, all species of poly(A)+ mRNA present in non-growing cells are also present in growing cells.

Nuclear RNA polymerase activities and poly(A)-containing mRNA accumulation in cultured AKR mouse embryo cells stimulated to proliferate.

Summary When non-growing AKR-2B mouse embryo cells are stimulated to proliferate by changing from serum-deficient to fresh media containing 10% serum, there is a consistent lag of 12 h before the onset of DNA synthesis. Endogenous DNA-dependent RNA polymerase activities, binding and initiation sites on isolated chromatin for exogenous RNA polymerase, and the rate of accumulation of poly(A)-containing polysomal RNA has been examined in AKR-2B cells with emphasis on the interval between stimulation and the onset of DNA synthesis. RNA polymerase type II activity, which is responsible for transcription of heterogeneous nuclear RNA (hnRNA), was increased by 1 h after stimulation and at 6 h reached peak levels which were 60–100% greater than the activity in resting cells. A rifampicin challenge method to assay for E. coli RNA polymerase binding and initiation sites on isolated chromatin was used to assay for changes in the amount of DNA available as a template for transcription. The results of this assay showed a slight decrease in the number of binding and initiation sites at 4 h followed by a slight increase at 6 h. The rate of accumulation of poly(A)-containing mRNA in polysomes showed a pattern and magnitude of increase following stimulation which was considerably different from that of RNA polymerase type II activity. There was a 4.5-fold increase over the resting levels by 2 h following stimulation. This enhanced level was maintained at all time points examined prior to the onset of DNA synthesis. These data suggest that both transcriptional and post-transcriptional mechanisms are responsible for the marked increase in polysomal poly(A)-containing mRNA observed after resting cells are stimulated to proliferate.

Gene expression in chemically transformed mouse embryo cells: Selective enhancement of the expression of C type RNA tumor virus genes

Abstract Treatment of a nontumorigenic clone of AKR mouse embryo cells in culture with a variety of polycyclic aromatic hydrocarbons has resulted in the development of derivative clones which are highly tumorigenic and exhibit other characteristics of the transformed phenotype. A 3-methylcholanthrene-transformed derivative clone (clone MCA) has been compared to the parent clone (clone 2B) with respect to the abundance and diversity of polysomal poly(A)-containing mRNA sequences. Hybridization kinetic experiments show that the poly(A)-containing sequences of both clones are organized into indistinguishable abundance classes, and that the vast majority of the sequences are common to both the parent and derivative clones. The levels of two specific messenger RNAs (α- and β-globin mRNA) which characterize highly differentiated mouse erythroid cells were much less than 1 molecule per cell in either cell type. Titration of a balanced complementary DNA probe to AKR murine leukemia virus (AKR-MuLV) 70S RNA with purified polysomal poly(A)-containing RNA from both parent and derivative clones shows that approximately 5000 and 1200 viral 35S RNA equivalents are present in the cytoplasm of growing and resting clone MCA cells, respectively. Rapidly growing clone 2B cells contain less than about 30 viral 35S RNA equivalents per cell. Viral specific sequences therefore correspond to members of the high abundance class of poly(A)-containing RNA sequences in clone MCA cells and to the low abundance class of sequences in clone 2B cells. Within the limits of detection, this large increase in abundance is characteristic only of viral specific RNA sequences.

Benzo(a)pyrene metabolism and blast transformation in peripheral blood mononuclear cells from smoking and nonsmoking populations and lung cancer patients.

Benzo(a)pyrene metabolism and lymphocyte transformation in peripheral blood mononuclear cells were evaluated in 3 groups of male patients. Group I were healthy nonsmokers, Group II were smokers, Group III were lung cancer patients, primarily stage I, evaluated before radiation or chemotherapy. Benzo(a)pyrene metabolism was assayed by a method involving quantitation of water soluble products produced from 3H‐benzo(a)pyrene over an eight hour reaction and lymphocyte transformation was measured by 3H‐thymidine incorporation. The mean level of metabolism of benzo(a)pyrene was significantly higher in the smoking control group, but was not significantly different in the nonsmoking control and the lung cancer groups. Lymphocyte transformation was significantly lower in the lung cancer patients than in either of the control groups despite the fact that 38 out of 57 of the lung cancer patients had stage I disease. Two pieces of evidence derived in this study indicate that the degree of lymphocyte transformation by mitogens influences the benzo(a)pyrene metabolism. First, the mean level of benzo(a)pyrene metabolites in lung cancer patients with lymphocyte stimulation less than 104 cpm was significantly lower than in those cancer patients with lymphocyte stimulation greater than 104 cpm. Secondly, when mononuclear cells from three control patients were stimulated with variable concentrations of mitogens, it was found that water soluble metabolite production and the degree of lymphocyte transformation had a significant correlation coefficient.

Age of meningitis or encephalitis is independently predictive of outcome from anterior temporal lobectomy.

Background: The occurrence of meningitis or encephalitis in early childhood, i.e., ≤4 years of age, may be associated with both the development of medial temporal lobe epilepsy (MTLE) and an excellent operative outcome following an anterior temporal lobectomy (ATL). However, whether the predictive value of this risk factor for partial epilepsy is independent of the finding of mesial temporal sclerosis (MTS) on MRI is not known. Methods: Consecutive patients (n = 39) with a remote history of meningitis or encephalitis who underwent an ATL were compared with 78 sex- and age-matched control subjects who had not experienced a CNS infection before ATL. All patients in both groups had nonlesional temporal lobe epilepsy and were followed up for at least 12 months postoperatively. Results: There was a trend for the patients with a history of meningitis or encephalitis to have a lower frequency of class I postoperative outcome (61.5% vs 73.1%, p = 0.21). In the meningitis or encephalitis group, a class I outcome was more frequent in those with a history of meningitis or encephalitis at a young age (<4 years) (19/23 vs 5/16, p = 0.002), those with MTS detected on a preoperative MRI (22/31 vs 2/8, p = 0.04), and those with a history of meningitis (16/21 vs 8/18, p = 0.05). Multivariate logistic regression analysis found that a history of meningitis or encephalitis at a young age (b = 2.0, O.R. = 7.5, p = 0.048) was predictive of a class I outcome independent of the presence of MRI-identified MTS (b = 2.0, O.R. = 7.3, p = 0.07). Conclusion: The age of occurrence of a remote history of meningitis or encephalitis, but not the type of infection, is predictive of outcome after an ATL independent of the finding of MTS on the preoperative MRI.

Aryl Hydrocarbon Hydroxylase in Man and Lung Cancer

Lung cancer is one of the leading causes of cancer deaths in most Western countries. In the United States it accounts for 33% of the cancer deaths in males and 11% of the cancer deaths in females. More males die from lung cancer than from the four next common cancer sites combined, i. e., colon, prostate, pancreas, and stomach cancer. In females, lung cancer holds third place in cancer deaths, preceded only by breast and colon cancer (Silverberg, 1977). Of all the common cancers it is the one most clearly associated with environmental agents, the most notable of which is cigarette smoking.

Transforming growth factor type β can act as a potent competence factor for AKR-2B cells

Transforming growth factor type beta (TGF beta) is a pleiotropic regulator of cell growth with specific high-affinity cell-surface receptors on a large number of cells; its mechanism of action, however, is poorly defined. In this report, we utilized the mouse fibroblast line AKR-2B to explore the question of the temporal requirements during the cell cycle in regard to both the growth inhibitory and the growth stimulatory action of TGF beta. The results indicate that AKR-2B cells are most sensitive to the inhibitory action of TGF beta during early to mid-G1. In addition, TGF beta need be present only briefly (as little as 1 min) in order to exert its inhibitory effect on EGF-induced DNA synthesis. Likewise, the stimulatory effect of TGF beta in the absence of EGF requires only an equally brief exposure to TGF beta. Use of homogeneous 125I-labeled TGF beta in a cell-binding assay demonstrates that TGF beta bound to cell-surface receptors can readily exchange into the culture medium T1/2 = 120 min), helping to rule out the possibility that persistent receptor-bound TGF beta is the source of a continuous stimulus. The data indicate that TGF beta exposure induces a stable state in the cell (T1/2 = 20 h) similar to but distinct from the state of competence induced by platelet-derived growth factor (PDGF).

Covalent binding of benzo[a]pyrene metabolites to DNA, RNA and chromatin proteins in the AKR mouse embryo cell-line

A specific fraction from the nuclei of the AKR mouse embryo cell-line (fraction I) displayed a much greater localization of radioactivity compared to fraction II and III when the chemical carcinogen, [3H]benzo[a]pyrene (B[a]P) was incubated with the cells for 24 h. The radioactivity in fraction I consisted of both covalently and non-covalently bound metabolites. Isolation of the DNA, RNA and protein of fraction I revealed that 94% of the covalently bound radioactivity was to protein, 5% to RNA and 1% to DNA. Analysis of the fraction I proteins by SDS gel electrophoresis revealed that there was more radioactivity covalently bound to the larger proteins than to smaller proteins. Isoelectric focusing (IEF) of the purified proteins displayed two peaks of radioactivity, one at a pH of 5 and the other at 11. The former proteins bound more radioactivity per mass of protein than the latter proteins. Analysis of fraction I histones on acid urea polyacrylamide gels showed that the radioactivity coincided with histones H3 and H2B and low levels of radioactivity associated with histones H1, H2A and H4. Two significant peaks of radioactivity closely migrated near but did not co-migrate with histone H1. The distribution of the bound radioactivity is probably a reflection of the availability of the proteins to the reactive carcinogen metabolites. The possible binding of B[a]P metabolites to phosphorylated histones and to the high mobility of group (HMG) proteins 1 and 2 is discussed.