Pharmacological inhibition of phosphatidylcholine biosynthesis is associated with induction of phosphatidylinositol accumulation and cytolysis of neoplastic cell lines.

De novo production of phosphatidic acid (PA) in tumor cells is required for phospholipid biosynthesis and growth of tumor cells. In addition, PA production by phospholipase D has been cited among the effects of certain oncogenes and growth factors. In this report, it has been demonstrated that enhanced phospholipid metabolism through PA in tumor cells can be exploited pharmacologically for development of anticancer agents, such as CT-2584, a cancer chemotherapeutic drug candidate currently in Phase II clinical trials. By inhibiting CTP:choline-phosphate cytidylyltransferase (CT), CT-2584 caused de novo phospholipid biosynthesis via PA to be shunted away from phosphatidylcholine (PC) and into phosphatidylinositol (PI), the latter of which was doubled in a variety of CT-2584-treated tumor cell lines. In contrast, cytotoxic concentrations of cisplatin did not induce accumulation of PI, indicating that PI elevation by CT-2584 was not a general consequence of chemotherapy-induced cell death. Consistent with this mechanism of action, propranolol, an inhibitor of PA phosphohydrolase and phosphatidylcholine biosynthesis, was also cytotoxic to tumor cell lines, induced PI accumulation, and potentiated the activity of CT-2584 in cytotoxicity assays. As expected from biophysical properties of anionic phospholipids on cellular membranes, CT-2584 cytotoxicity was associated with disruption and swelling of endoplasmic reticulum and mitochondria. We conclude that CT-2584 effects a novel mechanism of cytotoxicity to cancer cells, involving a specific modulation of phospholipid metabolism.

Estimation of sieving coefficients of convective absorption of drugs in perfused rat jejunum.

Intestinal absorption of many hydrophilic drugs cannot be explained solely in terms of pH-partition and solvent-drag effects have been described in a number of cases. However, quantitative estimates of sieving coefficient (phi) for drug molecules have tended to be variable. In the present work an in situ perfused intestinal loop preparation in the rat has been used to measure the disappearance of five hydrophilic drugs from the intestinal lumen and a mathematical model of drug absorption in the presence of net and unidirectional fluid fluxes has been developed. The model allows separate estimation of the convective (solvent drag) and nonconvective (partition) components of drug absorption from the experimental data. The five drugs studied were found to have phi values ranging from 0.1-0.9; this was highly dependent on molecular size. Analysis of the data shows that three of the drugs are absorbed almost exclusively by the convective process (caffeine, cimetidine, hydrochlorthiazide) while the other two are absorbed by both convective and nonconvective processes (salicylate, oxprenolol). We conclude that the methodology is a useful and reliable means of deriving separate estimates of these two components of drug absorption.

Association of pRas and pRaf-1 in a complex correlates with activation of a signal transduction pathway.

BACKGROUND: A key pathway for transduction of proliferative, developmental and oncogenic stimuli from receptors at the cell surface to transcription factors located in the nucleus involves the activation of pRas and pRaf-1. Recent publications have described a physical interaction between pRas and pRaf-1, either as ectopic proteins in yeast or as recombinant proteins added to cellular extracts. Until now, however, physical complexes that include pRas and pRaf-1 have not been identified as native structures in mammalian cells. RESULTS: We have directly identified a pRas-pRaf-1 complex in extracts of mammalian cells. Formation of the complex is augmented in neoplastically transformed cells expressing constitutively activated pRas. Moreover, the complexes form in concert with the activation of pRas during intracellular signalling through the T-cell receptor in T-leukemia cells. CONCLUSIONS: We propose that, pRas signals to pRaf-1 in vivo by means of a direct physical interaction that results in activation of the pRaf-1 protein kinase.

Predisposition to neoplastic transformation caused by gene replacement of H-ras1.

Homologous recombination was used to introduce a nominally transforming mutation into an endogenous H-ras1 gene in Rat1 fibroblasts. Although both the mutant and the remaining normal allele were expressed equally, the heterozygous cells were not neoplastically transformed. Instead, spontaneously transformed cells arose from the heterozygotes at a low frequency, and the majority of these cells had amplified the mutant allele. Thus, the activated H-ras1 allele was not by itself dominant over the normal allele but predisposed cells to transformation by independent events, such as amplification of the mutant allele.

A characterization of the preaggregative period of Dictyostelium discoideum.

The preaggregative period of Dictyostelium discoideum has been characterized by measuring the reduction in time for the onset of aggregation under conditions which hinder close cell-cell associations, inhibit protein synthesis, and/or include continuous high concentrations or pulsed low concentrations of exogenous cAMP. The results demonstrate that: the preaggregative period (normally 7 hr for cells from log phase cultures) can be dissected into two distinct components: an initial component which includes the first 4.5 hr, and a second component which includes the last 2.5 hr; the first component will progress at normal rate in the continuous absence of close cell-cell associations (as single amoebae in suspension) or in the continuous absence of de novo protein synthesis; the second component will not progress in the continuous absence of close cell-cell associations or de novo protein synthesis; high concentrations of cAMP continuously present in suspension cultures do not affect progress through the first component, nor do they support progress through the second component; however, if cells are allowed to form close cell-cell associations during progress through the first component, high concentrations of cAMP will support progress through the second component in the absence of close cell-cell associations; these associations, which render cells sensitive to cAMP, will occur in the absence of de novo protein synthesis and before the acquisition of contact sites A; these associations may be completely bypassed if suspended cells are continuously pulsed with low concentrations of cAMP; in this case, pulses of cAMP will support progress through the final component in continuous suspension cultures; and the acquisition of contact sites A will not occur in the absence of progress through the second component; in contrast, the acquisition of cAMP binding sites on the cell's surface will occur. These results are considered in terms of the complexity and regulation of the preaggregative period of Dictyostelium.

Regulation of protein synthesis during the preaggregative period of Dictyostelium discoideum development: involvement of close cell associations and cAMP.

The preaggregative period of Dictyostelium discoideum is composed of two rate-limiting components which exhibit dramatic differences in either their dependency upon, or sensitivity to, close cell-cell associations, inhibitors of protein synthesis, temperature, and pH. The first component comprises the initial 4.5 hr and the second component the last 2.5 hr of the preaggregative period. By pulse-labeling cells with [35S]methionine, separating polypeptides by 2D-PAGE, and semiquantitatively comparing the rates of synthesis of 778 individual polypeptides by fluorography, the following results were obtained: a detailed program of protein synthesis accompanies the preaggregative (0-7 hr) and aggregative (7-10.5 hr) periods of development; this includes significant decreases in the rate of synthesis of 93 polypeptides synthesized during vegetative growth and significant increases in the rate of synthesis of 74 polypeptides either undetectable or synthesized at relatively low rates during vegetative growth; 35 polypeptides are transiently synthesized at different times during the preaggregative and aggregative periods; two peaks of activity are clearly defined for both increases and decreases; these peaks correlate temporally with the first and second rate-limiting components of the preaggregative period; the majority of changes (74%) which occur during the first rate-limiting component will occur in the absence of close cell-cell associations, but the majority (66%) which normally occur during the second rate-limiting component do not occur in the absence of close cell-cell associations; a high concentration of cAMP in the medium of continuous suspension cultures does not stimulate most of the changes which are dependent upon close cell-cell associations; even though cAMP stimulates progress through the second rate-limiting component in suspension cultures first allowed to associate for 4.5 hr ("competent" cells) prior to disaggregation it still does not stimulate most of the changes which are dependent upon close cell-cell associations; and synthesis of only 3 out of 778 polypeptides appears to be stimulated by addition of exogenous cAMP, and only in resuspended cultures of "competent" cells. The prominent role of close cell-cell association and the surprisingly minor effect of cAMP in the regulation of the program of protein synthesis accompanying the preaggregative and aggregative periods of Dictyostelium are discussed, especially as they relate to the effect of cAMP on protein synthesis in suspended cultures of postaggregative cells.

Loss and resynthesis of a developmentally regulated membrane protein (gp80) during dedifferentiation and redifferentiation in Dictyostelium.

When developing cultures of Dictyostelium discoideum are disaggregated and resuspended in nutrient medium, they lose the capacity to rapidly reaggregate after 90 min, in a rapid and synchronous step referred to as the "erasure event." They then proceed to lose remaining developmentally acquired functions in a program of dedifferentiation culuminating with the loss of EDTA-resistant cohesion roughly 5 hr later. Immediately following the erasure event, cells can be stimulated to reenter the developmental program even though they still possess a number of developmentally acquired functions. These cells therefore appear to undergo dedifferentiation and redifferentiation simultaneously (D. R. Soll and L. H. Mitchell, 1982, Dev. Biol. 91, 183-190). In this report, we have employed an antiserum made against a developmentally acquired membrane glycoprotein, gp80, to examine whether gp80 is lost during dedifferentiation and whether it is either reutilized or resynthesized during redifferentiation. Results are presented which demonstrate that (1) when 9-hr developing cells are disaggregated and resuspended in nutrient medium, gp80 continues to accumulate for several hours after the erasure event, then is lost at roughly the same time as EDTA-resistant cohesion; (2) when cells are stimulated to reenter the developmental program immediately after the erasure event, both gp80 and EDTA-resistant cohesion are still lost according to the program of dedifferentiation, but are then reacquired soon afterwards according to the program of redifferentiation; (3) during redifferentiation, cells do not reutilize gp80 which had been synthesized during initial development; rather they synthesize gp80 de novo; and (4) developing cells of a dedifferentiation-defective variant, HI4, when disaggregated and resuspended in nutrient medium, retain gp80, EDTA-resistant cohesion, and the capacity to rapidly reinitiate aggregation for at least 12 hr. This last result indicates that the loss of gp80 is regulated by the dedifferentiation process and is not an independent response to disaggregation or the reintroduction of nutrients. Together, these results reinforce the conclusion that dedifferentiation and redifferentiation can function independently and simultaneously in the same cells.