Apoptosis of L929 cells by etoposide: a quantitative and kinetic approach.

Exponentially growing L929 cells were continuously exposed to 1 or 10 microM etoposide (VP-16). The effects of such treatment on cell growth, cycle distribution, morphology, and selected biochemical events were examined. DNA synthesis rates were markedly decreased and the protein/DNA ratio increased (unbalanced growth). Growth was blocked, with most cells being cycle arrested by 24 h in (late S-)G2-M. An asynchronous process of cell death then developed. Cells initially shrank into eosinophilic, trypan blue-excluding bodies, which were then released into the medium, and eventually became permeable to trypan blue. Transmission electron microscopy confirmed that dying cells acquired an apoptotic morphotype, with compaction and margination of chromatin, loss of microvilli, and shrinkage of cytoplasm and nucleus. Tissue transglutaminase activity and intensity of immunostaining rapidly increased in treated cultures. Internucleosomal DNA fragmentation could not be detected by agarose gel electrophoresis, yet flow cytometry revealed that the apoptotic bodies had a very low DNA fluorescence (< or = 10% of the 2n value). In agreement with the microscopic findings, this suggested that extensive DNA degradation had occurred in dead cells. While rates of cell loss from the monolayer amounted to 21 and 57% day(-1) (1 and 10 microM VP-16, respectively), apoptotic indexes largely underestimated the extent of the process. These indexes only measured the accumulation of apoptotic bodies, i.e., the balance between their generation and disposal. The latter occurred by mechanisms similar to those that operate in tissues: "secondary necrosis" or phagocytosis by viable homotypic cells in the monolayer ("homophagy").

Intracellular ionic variations in the apoptotic death of L cells by inhibitors of cell cycle progression.

Treatment with VP-16 (1-50 microM) or excess thymidine (5 mM) caused a block of L cells at different steps in their progression through the replicative cycle. The arrest was followed by an asynchronous process of cell death that conformed to criteria for apoptosis. Careful monitoring of this process in the whole cell population by flow cytometry showed a virtual absence of necrosis, an increase in side light scattering, followed by the occurrence of a population with subdiploid DNA fluorescence as well as reduced forward and side light scattering. The development of apoptosis required sufficient time and adequate ion gradients in the cells. By the combined use of flow cytometry and fluorescence microscopy data were obtained suggesting that (i) intracellular free Ca2+ and pH and/or their drug-induced alterations had to be adequately controlled for the apoptotic process to evolve; (ii) mitochondria were compromised earlier than the plasma membrane or lysosomes; and (iii) K+ extrusion possibly played a role in the final loss of cell volume. Interfering with the control of ion gradients and/or their changes in drug-treated cells resulted in cell death by necrosis.

Diagnostic power of lecithin/sphingomyelin ratio and fluorescence polarization assays for respiratory distress syndrome compared by relative operating characteristic curves.

Amniotic fluids from 328 patients were analyzed for lecithin/sphingomyelin (L/S) ratio and surfactant/albumin (S/A) ratio by fluorescence polarization. Of this group, 61 neonates showed respiratory distress syndrome (RDS) on delivery within 3 days of testing. We compared the power of the L/S and S/A in diagnosing pulmonary maturity, using relative operating characteristic (ROC) curves. The area defined by the ROC curve of the S/A test exceeded the area defined by the L/S curve, but this difference was not statistically significant. The diagnostic power of the S/A test appears to be at least equal to that of the standard L/S test. A review of five cases of RDS in which laboratory tests had suggested maturity showed that neither the L/S nor the S/A could satisfactorily resolve the problem of false interpretations of maturity, particularly in mothers with diabetes mellitus who underwent cesarean section.

Cell death induced in L-cells by treatment with thymidine: staging of the process and relationship to apoptosis.

The purpose of this study was to characterize the stages in the development of thymidine-induced cell death. L-cells were characterized by both morphologic and quantitative techniques and evaluated at 24, 48, and 72 h of treatment. Cells first enlarged (stage I); about 50% of these enlarged cells then decreased in size with blebbing and compacting (stage II). This residual cell body transformed into a smooth eosinophilic hyaline body (stage III) by 72 h, many of which could be identified within the vacuolar system of viable cells. These changes were reflected in morphologic counts and Coulter sizing. Cell death (loss of labeled DNA) began in stage II and was most prominent in stage III. No cleavage of DNA into oligonucleosomal fragments was detected by agarose gel electrophoresis at any stage. The similarity of these changes to the complete spectrum of apoptosis in vivo is discussed.

Estrogen receptors and the response to sex hormones in angiolymphoid hyperplasia with eosinophilia.

BACKGROUND: Angiolymphoid hyperplasia (ALH) with eosinophilia is a benign rare tumor, characterized by marked proliferation of endothelial cells. The tumors are associated with extensive infiltrate of lymphocytes, histiocytes, and eosinophils, and occur on the head and neck of young adults. A variety of treatments have been attempted with frequent recurrences. We report two cases of ALH with eosinophilia that seemed to be dependent on sex hormones. OBSERVATION: The first case is a patient with ALH that resolved after stopping treatment with birth control pills. Biopsy specimens of the tumor demonstrated increased level of estrogen and progesterone receptors compared with her normal skin. The second case is a patient with a previous lesion of ALH with eosinophilia, who during pregnancy had new lesions develop and whose primary lesion increased in size. All lesions decreased to half their original sizes after pregnancy. CONCLUSION: Both of these cases suggest a role for hyperestrogen states with the presence of hormonal receptors. The response to sex hormones could contribute to the pathogenesis of the disease and may offer future alternative treatment modalities.

Proteolysis and cell death.

Cell death can occur by a number of different mechanisms, some of which are associated with significant protein degradation. L-cells in monolayer culture were labelled with 14C-leucine and 3H-thymidine and placed in a cold chase medium for each experiment. General cellular necrosis, induced by either repeated freeze-thawing or NaCN, rapidly disrupted cellular structure, but produced only small amounts of acid soluble 14C over a period of 24 h. Selective cell death was produced by adding 5 mM thymidine to growing tissue cultures. With such treatment, cell death became manifest only after a 24 h lag period and progressively more intense in the next 48 h. The process was characterized first by cellular enlargement and protein accumulation, followed by the formation and release of eosinophilic hyaline bodies from cell cytoplasm. These bodies were often found adjacent to the plasma membranes of viable cells, as well as within cellular vacuoles. Biochemical studies indicated that proteolysis was significantly increased; this proteolysis, but not cell death, was prevented by NH4Cl. Cell death, formation of eosinophilic hyaline bodies, and proteolysis were prevented by cycloheximide. Cell cultures that manifest cell turnover, i.e. selective cell death, apoptosis-type, will show increased amounts of vacuolar proteolysis, which may be confused with accelerated protein turnover occurring in the vacuolar system.

Lipid composition and de novo cholesterogenesis in normal and neoplastic rat mammary tissues.

Female Wistar rats were given 5 mg of 7,12-dimethylbenz[a]anthracene (DMBA) and fed either a control diet (AIN), a 4% cholestyramine (CHST) diet, a 2% corn oil plus 18% coconut oil (saturated fat) diet, a 20% corn oil [unsaturated fatty acid (USF)] diet, or a USF + 4% cholestyramine (USF + CHST) diet. The mammary glands, tumors, livers, and sera were analyzed for lipids, de novo cholesterogenesis, and serum lecithin-cholesterol acyltransferase (LCAT) activity levels. Level and type of fat in the diet, DMBA, CHST, and length of feeding influenced the lipid composition of liver and mammary tissues. Stimulation of de novo cholesterogenesis in the mammary gland and depression in circulating LCAT activity levels correlated with the incidence and growth of mammary tumors, suggesting that stimulation of de novo cholesterogenesis plays an important role in mammary cancer development.

Regulation of protein turnover versus growth state. Studies on the mechanism(s) of initiation of acidic vacuolar proteolysis in cells of stationary ascites hepatoma.

1. After transplantation, the rat AH-130 Yoshida ascites hepatoma enters a phase of exponential (log) growth, followed by a quasi-stationary (sta) state. Combining measurements made in vivo and in vitro, cessation of protein accumulation (growth) in sta phase has previously been shown to result from convergent reduction of protein synthesis and enhancement of protein breakdown [Tessitore, Bonelli, Cecchini, Amenta & Baccino (1987) Arch. Biochem. Biophys. 255, 372-384]. 2. One day after labelling in the animal with [3H]leucine, AH-130 cells were processed for short-term assays in vitro to measure rates of endogenous protein breakdown. 3. Exposure of AH-130 cells to inhibitors interfering with different steps of the acidic vacuolar pathway (AVP) showed that: (i) in log tumour cells the AVP was extensively suppressed; (ii) in sta tumour cells virtually all of the proteolytic acceleration was accounted for by activation of the AVP. 4. Treating log tumour cells with glucagon, cyclic AMP, or nutritional deprivation failed to elevate substantially the proteolytic rates. Nor could the elevation in proteolysis be explained by changes in free amino acids, which were more concentrated in the ascitic fluid of sta tumours. 5. The enhanced proteolysis in sta tumour cells was not associated with any increase in the intracellular activity levels of lysosomal cathepsins B, D, H, and L. 6. The above growth-related modulation of protein breakdown in AH-130 cells was probably a reflection of the tumour growth state rather than the direct effect of environmental stimuli.

Comparing different statistical methods for evaluating diagnostic effectiveness of clinical tests: respiratory distress syndrome as a model.

Phospholipids in specimens of amniotic fluid from 346 patients were quantified and the results evaluated in light of the clinical outcome. Fifty-eight neonates had respiratory distress syndrome. We used this data base to compare different statistical methods for evaluating test effectiveness and diagnostic discrimination. Dichotomizing quantitative tests into binary tests with arbitrary cutoff values was inadequate for comparing test effectiveness. Subgrouping the data into deciles and calculating the incidence of respiratory distress syndrome for each decile avoided the problems of the preceding approach and was easy to calculate and comprehend; however, this method lacked statistical power. Relative operating characteristic curves yielded more statistical power, but results were more difficult to calculate and were not intuitively obvious to most workers in the laboratory. A modified cumulative frequency plot, combining elements of both decile subgrouping and relative operating characteristic curves, was easily calculated and intuitively obvious. These plots, like relative operating characteristic curves, provided an index for quantifying test effectiveness. When used in combination with standard cumulative frequency curves, they also provided direct diagnostic information on disease probability for any value of the clinical assay.

Regulation of protein turnover versus growth state: ascites hepatoma as a model for studies both in the animal and in vitro.

Cell protein turnover states as related to growth phase have been analyzed in a rat ascites hepatoma (Yoshida AH-130), which after transplantation entered a period of exponential growth, followed by a quasi-stationary state. Evaluation of AH-130 cell protein turnover in the animal (slow-turnover protein pool) was combined with rapid assays of proteolytic rates of cells transferred in vitro. Protein accumulation in the exponential phase reflected the balance between sustained synthetic rates and relatively low degradative rates. Cessation of growth resulted from convergent reduction of synthesis (from 3.10 to 1.49%/h) and enhancement of protein breakdown (from 0.61 to 1.43%/h). Endogenous proteolytic rates in vitro were very close to the above degradation rates. As shown by incubation with ammonia or other lysosomal inhibitors, the acidic vacuolar pathway for protein degradation, while totally suppressed in exponential tumor cells, was activated in cells from stationary tumors to such an extent that it fully accounted for the enhanced proteolysis. In contrast, energy metabolism inhibitors were effective on cells in either growth state, the residual ongoing proteolysis being similar in both cells. The possible contribution of cell death to activation of the acidic vacuolar proteolysis in stationary tumors is discussed.

Evaluating the clinical effectiveness of amniotic fluid assays in predicting respiratory distress syndrome in the neonate.

Amniotic fluid phospholipids from 346 patients' specimens were quantified and evaluated against the clinical outcome (i.e., respiratory distress syndrome or normal respiratory function). Concentrations of lecithin, sphingomyelin, phosphatidylglycerol, and the lecithin/sphingomyelin reflectance ratio were evaluated by ordered frequency distribution and stepwise discriminant function analysis. The lecithin/sphingomyelin ratio was the best single test for discriminating between respiratory distress syndrome and normal pulmonary function in the fetus, slightly superior to lecithin assay alone. A combination of lecithin/sphingomyelin ratio and lecithin concentration, however, appeared to optimize the discriminant function, although the clinical significance of this test combination remained marginal. High concentrations of phosphatidylglycerol were correlated with high concentrations of lecithin, and virtually ruled out respiratory distress syndrome. Absence of phosphatidylglycerol was not diagnostic. High concentrations of sphingomyelin increased the probability of respiratory distress syndrome. We suggest the following stepwise series of tests to optimize diagnosis: phosphatidylglycerol concentration, sphingomyelin concentration, and finally lecithin-sphingomyelin ratio.

Evidence for a special relationship between proteolysis and single cell necrosis.

A high rate of single cell necrosis is a common phenomenon in neoplastic and preneoplastic lesions, accounting for growth rates that are significantly less than the cell birth rate. We present data relating the process of protein turnover to single cell necrosis. Cells were labeled with 3H-leucine and 14C-thymidine; the loss of radioactivity from the cell protein and DNA was then measured for 3-6 days. Preliminary experiments showed that cell necrosis by freeze-thawing cells did not significantly contribute to the degradation of cell proteins. Similar results were observed with dying 3T3-SV40 cells at high density. L-cells, however, showed a progressive increase in cell loss as higher cell densities were attained on the monolayer. Although proteolysis remained constant in the culture, analysis of the cells recovered from the high density monolayers showed little loss of labeled protein after adjustment for loss of label in the DNA. Three possible explanations are proposed: DNA turns over with cell protein (unlikely), single cell necrosis involves a special mechanism that facilitates reutilization of amino acids, or single cell necrosis includes only cells that are selectively involved in protein turnover. A unique relationship between single cell necrosis and proteolysis is suggested.

Analytical errors in measuring radioactivity in cell proteins and their effect on estimates of protein turnover in L cells.

Previous studies from this laboratory on protein turnover in 3H-labelled L-cell cultures have shown recovery of total 3H at the end of a 3-day experiment to be always significantly in excess of the 3H recovered at the beginning of the experiment. In this study we have critically reviewed a number of possible sources for this error in measuring radioactivity in cell proteins. 3H-labelled proteins, when dissolved in 0.3 M-NaOH and counted for radioactivity in a liquid-scintillation spectrometer, showed losses of 30-40% of the radioactivity; neither external or internal standardization compensated for this loss. Hydrolysis of these proteins with either Pronase or concentrated HCl significantly increased the measured radioactivity. In addition, approx. 5-10% of the cell protein is left on the plastic culture dish when cells are recovered in phosphate-buffered saline. To aggravate this latter loss further, this surface-adherent protein, after pulse labelling, contains proteins of high radioactivity that turn over rapidly and make a major contribution to the accumulating radioactivity in the medium. These combined errors can account for up to 60% of the total radioactivity in the cell culture. Similar analytical errors have been found in studies of other cell cultures. The effect of these analytical errors on estimates of protein turnover in cell cultures is discussed.

Intracellular degradative fate of transplanted and microinjected proteins.

The mechanisms of intracellular protein catabolism have not been elucidated in spite of 42 years since Schoenheimer's treatise on "The Dynamic State of Body Constituents". Protein catabolism (cf. Protein Synthesis) involves multiple mechanisms, several of which occur reliably only in cells, not test-tubes. Elucidation of the mechanisms relies on both cell biological and enzymological approaches. Transplantation and microinjection of proteins into target cells, although naturally not without experimental and interpretive difficulties, provide the means of identifying the molecular and cell biological events in intracellular protein degradation. In short, these techniques provide the bioassay system to probe the functions of known catalysts and inhibitors of proteinolysis and provide the means of identifying the currently unknown features of the cytoplasmic surveillance system which present the protein substrates for destruction.

Sources of error in estimating radioactivity in protein from cell cultures by liquid scintillation counting.

In this study, several common sample preparation techniques for liquid scintillation counting were critically reviewed. It has been shown that techniques such as NaOH or formic acid solubilization of trichloroacetic acid (TCA)-precipitated proteins led to underestimation of the radioactivity by 20-40%; this loss was not corrected by either internal or external standardization. Hydrolysis of the proteins with 6 N HCl or Pronase significantly increased the recovery of the labeled proteins. Also, 10% of the labeled cell proteins remained on the dish when cells were scraped into buffer; these labelled proteins could be recovered either by in situ hydrolysis with Pronase or by solubilization and scraping in 0.3 N NaOH. These techniques increased the recovered radioactivity by 50-60%, allowing quantitative measurements to be made over a 3-day chase period. A possible mechanism and the implications of this observation were discussed.

Degradation of proteins in rat liver mitochondrial outer membrane transplanted into different cell types. Evidence for alternative processing.

The degradation of proteins in reductively [3H]methylated mitochondrial outer membrane (MOM) transplanted into cells by a poly(ethylene glycol)-mediated process has been studied. The average rate of degradation (t1/2 24-28 h) of MOM proteins transplanted into HTC cells was not the same as for endogenous MOM proteins (t1/2 56 h), mitoplast proteins (t1/2 120 h), plasma membrane proteins (t1/2 approx. 90 h) or cytosol proteins (t1/2 75 h). The degradation of transplanted MOM proteins was inhibited to the same extent (30-45%) as that of endogenous mitochondrial and plasma membrane proteins by leupeptin and NH4Cl. No inhibition of HTC cell cytosol protein degradation by NH4Cl was observed. NH4Cl differentially inhibited the degradation of endogenous MOM and mitoplast protein subunits as shown after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Proteins in MOM transplanted into tissue culture cells were degraded either with t1/2 24-28 h (MRC-5, B82 and A549 cells) or with t1/2 55-70 h (CHO-K1 and 3T3-L1 cells) similar to that of proteins in MOM transplanted into rat hepatocytes [Evans & Mayer (1983) Biochem. J. 216, 151-161]. The data suggest that membrane protein destruction is but the end part of a fundamental intracellular membrane recognition process.

Deficient lung surfactant apoproteins in amniotic fluid with mature phospholipid profile from diabetic pregnancies.

An enzyme-linked immunoassay to quantitate lung surfactant apoproteins (15 to 250 ng/ml) in human amniotic fluid is described. The immunoassay was used to quantify lung surfactant in 72 samples of amniotic fluid, for which lecithin/sphingomyelin (L/S) ratios, lecithin and phosphatidylglycerol concentrations, and foam stability indices were also available. The results obtained with the immunoassay were in general agreement with those of the other methods. Measurement of the apoproteins, however, may be a better predictor of fetal lung immaturity and of respiratory distress syndrome than the L/S ratio and the concentration of lecithin. This conclusion is based on the data obtained in the analyses of samples of amniotic fluid from four diabetic and five nondiabetic pregnancies, the infants of which developed respiratory distress. In all cases, the apoprotein concentration was less than 2.1 micrograms/ml, which indicated fetal lung immaturity. In six of these cases (one diabetic and five nondiabetic pregnancies), lung immaturity was also predicted on the basis of other tests. However, in three other cases of diabetic pregnancy, the L/S ratio and lecithin concentration falsely indicated lung maturity. In addition to its being an effective predictor of fetal lung maturity in diabetic, as well as nondiabetic, pregnancies, the immunoassay is better suited for clinical use because of its high specificity, sensitivity, and ease of performance.