The isolation of microsomal subfractions of mouse plasmacytoma cells: The effect of salt concentration during nitrogen cavitation

Following disruption of MPC-11 cells by nitrogen cavitation the microsomes have been fractionated by centrifugation on discontinuous sucrose gradients. When the homogenization buffer contained 25 mm KCl three fractions were observed: smooth microsomes, light rough microsomes, and heavy rough microsomes. When it contained 100 mm KCl, however, only smooth and light microsomal fractions were found. Under the latter conditions the heavy rough microsomal vesicles were apparently not released as separate organelles but instead sedimented together with the endoplasmic reticulum which remains attached to the nuclei.     

Isolation of intracisternal type A-particles and associated high-molecular-weight RNA after cell disruption by nitrogen cavitation.

The liberation of intracisternal A-type particles from the endoplasmic reticulum of mouse plasmacytoma cells (line MPC-11) without the use of detergents, has been achieved by the technique of nitrogen cavitation. By this method cell rupture is due to a sudden decompression of a cell suspension after being equilibrated with nitrogen at high pressure, and this results in a relatively efficient release of A-type particles from the cisternae as judged by electron microscopy and the amount of A-particle-associated RNA that may be isolated. Comparative studies showed that at least 50% of the particles are released from the endoplasmic reticulum during nitrogen cavitation. Since the release is achieved purely by mechanical means, the A-particle preparation obtained may be useful for assaying these particles for biological activity. A further release of particles from the endoplasmic reticulum may be achieved by a combination of detergent treatment and mechanical shearing. About 30% of the particle-associated RNA was polyadenylated. As determined by velocity centrifugation, the polyadenylated RNA in the preparation of A-particles released during nitrogen cavitation and that of the detergent-released A-particles were similar with respect to the high-molecular-weight RNA species present. A dominant 35 S RNA species with a 30 S shoulder was observed in addition to a 24 S component in both preparations, but no significant amount of 60–70 S RNA.     

Chemical and immunological studies of cell surfaces from normal and transformed cells

Immunological and chemical studies of cell surfaces from normal and transformed BALB/c fibroblasts have shown alterations associated with transformation. The cells studied include normal lines which do not cause tumors when injected into BALB/c mice, viral transformants, and spontaneous transformants which cause tumors that either regress or grow progressively, killing the host. The spontaneously transformed progressors include cell lines which are immunogenic and nonimmunogenic as determined by the ability of tumor excision to protect an animal from subsequent rechallenge by tumor cells. Tumor-bearing mice produce lymphocytes which are nonspecifically cytotoxic for all the normal and transformed lines. Some of the cell lines induce specific antibody formation in BALB/c hosts. Antisera have been prepared in rabbits which are specific for the transformed cell lines. These antisera can be used to determine specific surface changes on the transformed cells. Chemical studies have shown glycolipid alterations between the normal cells and some, but not all, of the transformants. Glycoproteins labeled by lactoperoxidase-¹²⁵ I or [³H] glucosamine were compared by SDS gel electrophoresis. Results from these studies do not show changes associated with malignancy. Individual glycoprotein regions from gels were treated with pronase, and the glycopeptides compared by Sephadex G-50 chromatography. Alterations in glycopeptides from several cellular glycoproteins are the only changes which appear to be associated with malignancy.     

TRP channels in cell survival and cell death in normal and transformed cells

TRP channels form a superfamily of channel proteins exhibiting versatile regulatory characteristics with many channels participating in the regulation of Ca²⁺ homeostasis and influencing the cell fate. Multitude of evidence is emerging that the colocalization of TRP channels with Ca²⁺-sensing elements of specific regulatory pathways leading to either proliferation or apoptosis is what makes these channels participate in cell fate regulation and, in turn, determines the final effect of Ca²⁺ entry via the particular channel. This review focuses on the aspects of TRP channel localization and function that affect the balance between cell survival and death and how various dysregulations of these channels may lead to perturbed balance and onset of cancer.     

Malondialdehyde production from spermine by homogenates of normal and transformed cells

The oxidation of spermine in vitro by a mixture of polyamine oxidase and diamine oxidase from pig kidney gives rise to malondialdehyde via 3-aminopropanol as the intermediate. Conversely, with spermidine, under similar experimental conditions, no evidence could be obtained for malondialdehyde formation within the limits of sensitivity of the assay (2.0 nmol). The activities of both these enzymes show about a 2-fold increase in normal rat kidney cells (LA31 NRK) transformed by the temperature sensitive mutant of Rous sarcoma virus (LA31) and incubated at the non permissive temperature (39 degrees C) compared to the activities in LA31 NRK at the permissive temperature (33 degrees C). These same enzymatic activities show no temperature dependent changes in normal rat kidney cells (NRK) or in these same cells infected by the wild type virus (NRK B77). In extracts derived from Friend erythroleukemic cells induced to differentiate by dimethyl sulfoxide or hexamethylene bis acetamide, spermine oxidation takes place more efficiently than in non induced cells. A rise in diamine oxidase activity is seen in LA31 NRK (39 degrees C) 12 h after the temperature shift, whereas morphological manifestations of normalcy are seen only at 48 h. The Km of diamine oxidase is 10(-6) M for putrescine and 10(-3) M for 3-aminopropanol. A possible mechanism involving the well documented acetylation of putrescine [23,26] is proposed for diverting intracellular putrescine away from cytosolic diamine oxidase and towards intramitochondrial monoamine oxidase.     

Changes in the surface morphology of normal and transformed mouse fibroblasts following disruption of cell-substrate adhesion]

By means of scanning electron microscopy surface morphology of cultured normal mouse embryo fibroblasts (MEF) and transformed mouse fibroblasts of L strain was studied in the course of alteration of cell-substrate adhesion with proteases, EDTA and urea. The morphology of cell rounding induced by the above agents in MEF and L cells was almost independent on the type of the agent. The rounding of MEF proceeded through three stages and was accompanied by substantial changes of cell surface relief. L cells lacked the intermediate stage (formation of thick processes) during their rounding which proceeded without any changes of cell surface relief. It is suggested that the observed differences are related to the poorer development of the lamelloplasm and microfilaments bundles in the transformed cells ascompared to the normal ones.     

Secretion of RNA by normal and transformed cells

3T3 and SV-40 transformed 3T3 cells in culture secrete RNA into their culture media. This medium RNA is predominantly 5s in size as measured by sucrose gradient and Sephadex gel filtration. Medium RNA is metabolically stable and is heavily methylated in comparison with other major cytoplasmic species. Analysis of the radioactively labeled methylated bases of medium RNA by paper chromatography after formic acid hydrolysis shows a very simple pattern of two peaks in contrast to the very complex patterns seen in rRNA and tRNA. Mycoplasma and mouse leukemia virus contamination have been excluded. The source of this RNA is discussed.     

Structural comparison of fibronectins from normal and transformed cells

Comparative study of the structures of fibronectins from normal and transformed cells by partial proteolysis as well as by tryptic peptide fingerprinting and analysis of phosphorylation show that: 1) fibronectin molecules from normal and transformed cells probably have very similar primary structures; 2) the phosphorylation of fibronectin is a highly specific and conserved phenomenon; 3) fibronectin from both normal and transformed cells is phosphorylated only on serine residues; 4) although the major sites of phosphorylation in fibronectin are the same in normal and transformed cells, fibronectin from transformed cells appears to be phosphorylated to a much higher extent than that from normal cells.     

The centrosome in normal and transformed cells

The centrosome is a unique organelle that functions as the microtubule organizing center in most animal cells. During cell division, the centrosomes form the poles of the bipolar mitotic spindle. In addition, the centrosomes are also needed for cytokinesis. Each mammalian somatic cell typically contains one centrosome, which is duplicated in coordination with DNA replication. Just like the chromosomes, the centrosome is precisely reproduced once and only once during each cell cycle. However, it remains a mystery how this protein-based structure undergoes accurate duplication in a semiconservative manner. Intriguingly, amplification of the centrosome has been found in numerous forms of cancers. Cells with multiple centrosomes tend to form multipolar spindles, which result in abnormal chromosome segregation during mitosis. It has therefore been postulated that centrosome aberration may compromise the fidelity of cell division and cause chromosome instability. Here we review the current understanding of how the centrosome is assembled and duplicated. We also discuss the possible mechanisms by which centrosome abnormality contributes to the development of malignant phenotype.     

Proteolytic enzymes in normal and transformed cells

Virally transformed cells show an increased production of proteolytic enzymes. These might be involved in transformation-dependent alterations of cell surface glycoproteins. The possibility arises that some of these proteases might be membrane-bound. To investigate this possibility, we have undertaken a comparative study of the reactivity of intact normal and transformed cells with the tritium labelled protease inhibitor diisopropylfluorophosphate, in parallel with fibrinolytic assays. Using these two approaches in concert, it was possible to identify and localize in the transformed cells several proteases which were present in the particulate cell fraction and were probably membrane bound. In particular, a diisopropylfluorophosphate-reactive polypeptide of 62 000 was increased 5–8-fold on transformation. It comigrated with a fibrinolytic activity. Other particle-bound activities were also detected. While diisopropylfluorophosphate-labelling can be useful for detecting proteases inside cells, it does not appear to be specific for surface proteases.     

Pyrimidine biosynthesis in normal and transformed cells

We have developed procedures for sensitive measurement of specific radioactivities of pyrimidine nucleosides excreted from cells in culture. The changes in the observed values reflect dilution of the added isotope through de novo biosynthesis of nonradioactive pyrimidine nucleosides or by shifting and equilibration of other nucleotide pools into the free uridine pool. It is thus possible to monitor uridine biosynthesis occurring in intact cells without destroying or disrupting the cell population. On comparing a series of normal and transformed lines, we have observed several growth-dependent patterns of change in specific activity and levels of uridine excretion and the temporal appearance of these changes. Hamster embyro fibroblasts slows pyrimidine biosynthesis at mid-growth while the hamster cell line V79 continues to dilute the pyrimidine pool at about 7% of the rate observed during exponential growth at confluence. Both cells exhibit Urd excretion beginning at one-half maximal growth. Passageable normal rat liver cells (IARC-20) also show a cessation of pyrimidine biosynthesis with a prior increase in uridine excretion. Two chemically transformed lines IARC-28 and IARC-19 derived from IARC-20 show different patterns. IARC-19 begins uridine excretion in early log growth and the specific activity continues to decrease at about 2% of the rate observed during exponential growth at confluence. The IARC-28 cells also begin excretion in early log growth but pyrimidine biosynthesis stops at about midlog. This method may prove to be an additional aid in recognizing and differentiating transformed cells in culture that do not exhibit the transformed phenotype.     

Differences in DNA-binding proteins isolated from normal and transformed human cells

When the DNA-binding proteins (DBPs) of WI38 normal human fibroblasts and their SV40-transformed counterpart were compared, two DBPs were present in greater amounts in the transformed cells. These two DBPs, P5a and P6b, were also present in greater amounts in HeLa cells versus WI38 cells and in chemically transformed human liver cells versus normal liver cells. Therefore, these DBP differences do not appear to be specific for transformation by SV40. Increased amounts of P5a were present in 7 of 9 transformed cell lines examined. The two tumor cell lines lacking the P5a change were sensitive to density-dependent inhibition of replication, whereas the other seven cell lines were not. This correlation suggests that the increase in P5a may play a role in the release from density-dependent inhibition of replication observed in most transformed cells.     

Hypertensive factor: calcium stimulatory activity obtained from different tissues and animal species

It was recently shown that a peptide (hypertensive factor, HF) isolated from erythrocyte hemolysates from spontaneously hypertensive rats induced a prolonged elevation of blood pressure in normotensive rats. In addition, the peptide produced a marked stimulation of the in vitro uptake of lanthanum-resistant calcium by the aortae and enhanced the contractile response of aortic rings to constrictor agents. The present report describes findings of calcium stimulatory activity, enhancement of contractile function, or pressor activity in extracts of homogenates from several tissues of the rat and from erythrocyte hemolysates of several mammalian species. Significant stimulation of calcium uptake in aortic rings was obtained with preparations from rat brain, liver, and kidney. The activity per weight of tissue was similar for brain and kidney (approximately 2 units/g), while liver exhibited somewhat higher concentrations (4 units/g). The diffusate of cardiac tissue did not significantly alter in vitro calcium uptake by aortae. The injection of the cardiac and liver diffusates into normotensive Wistar-Kyoto rats produced slight (10 Torr) (1 Torr = 133.3Pa) and moderate (25 Torr) elevations of blood pressure, respectively. Finally, a peptide purified from homogenates of rat brain by the protocol developed for the purification of HF from erythrocytes was shown to significantly enhance the contractile response of aortic rings to K+ and norepinephrine. Diffusates of erythrocytes from the rat, rabbit, dog, and guinea pig each caused a significant stimulation of calcium uptake and contained approximately the same level of activity (500 units/L of whole blood). Diffusates prepared from outdated human erythrocytes had no significant effect on calcium uptake, whereas those of freshly drawn samples exhibited high levels of activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-ion-transporting activity in two microsomal subfractions from rat pancreatic acini. Modulation by carbamylcholine

Two microsomal subfractions from isolated rat pancreatic acini were produced by centrifugation through a discontinuous sucrose density gradient and characterized by biochemical markers. The denser fraction ( SF2 ) was a highly purified preparation of rough endoplasmic reticulum; the less-dense fraction ( SF1 ) was heterogeneous and contained Golgi, endoplasmic reticulum and plasma membranes. 45Ca2+ accumulation in the presence of ATP and its rapid release after treatment with the bivalent-cation ionophore A23187 were demonstrated in both fractions. The pH optimum for active 45Ca2+ uptake was approx. 6.8 for the rough endoplasmic reticulum ( SF2 ) and approx. 7.5 for SF1 . Initial rate measurements were used to determine the affinity of the rough-endoplasmic-reticulum uptake system for free Ca2+. An apparent Km of 0.16 +/- 0.06 microM and Vmax. of 21.5 +/- 5.6 nmol of Ca2+/min per mg of protein were obtained. 45Ca2+ uptake by SF1 was less sensitive to Ca2+, half-maximal uptake occurring at 1-2 microM-free Ca2+. When fractions were prepared from isolated acini stimulated with 3 microM-carbamylcholine, 45Ca2+ uptake was increased in the rough endoplasmic reticulum. The increased uptake was due to a higher Vmax. with no significant change in Km. No effect was observed on 45Ca2+ uptake by SF1 . In conclusion, two distinct non-mitochondrial, ATP-dependent calcium-uptake systems have been demonstrated in rat pancreatic acini. One of these is located in the rough endoplasmic reticulum, but the precise location of the other has not been determined. We have shown that the Ca2+-transporting activity in the rough endoplasmic reticulum may have an important role in maintaining the cytosolic free Ca2+ concentration in resting acinar cells and is involved in Ca2+ movements which occur during stimulation of enzyme secretion.     

Phenotypes of cell hybrids formed by fusion of normal and Rous sarcoma virus transformed cells

Untransformed mouse cells were fused with rat cells transformed by a temperature sensitive mutant of avian sarcoma virus, and cell hybrids were isolated in the absence and in the presence of selective medium. None of the hybrids isolated were as transformed as the parent rat cells. All hybrids isolated in the absence of selective medium showed a phenotype similar to that of the untransformed mouse cell parent. Cell hybrids isolated on selective media, however, were more heterogenous. Some showed a phenotype that were intermediate between that of the two parental cells, while others were more like the untransformed mouse cells.