Growth control of normal and transformed cells

Both serum factors and protein synthesis are required for normal cell growth. Swiss 3T3 cells require the serum growth factors insulin and EGF (epidermal growth factor) during the initial part of the G₁ period, until they pass a restriction point about 2 h before the initiation of DNA synthesis. Concentration of cycloheximide that inhibit protein synthesis by as much as 70% dramatically lengthen the cell cycle before the restriction point, while the cell cycle after the restriction point remains nearly constant. These results are consistent with a model in which labile proteins are required for transit of cells past the serum-sensitive restriction point. The relation of these findings to the growth control of transformed cells is discussed.     

Growth control mechanisms in normal and transformed intestinal cells.

The cells populating the intestinal crypts are part of a dynamic tissue system which involves the self-renewal of stem cells, a commitment to proliferation, lineage-specific differentiation, movement and cell death. Our knowledge of these processes is limited, but even now there are important clues to the nature of the regulatory systems, and these clues are leading to a better understanding of intestinal cancers. Few intestinal-specific markers have been described; however, homeobox genes such as cdx-2 appear to be important for morphogenic events in the intestine. There are several intestinal cell surface proteins such as the A33 antigen which have been used as targets for immunotherapy. Many regulatory cytokines (lymphokines or growth factors) influence intestinal development: enteroglucagon, IL-2, FGF, EGF family members. In conjunction with cell-cell contact and/or ECM, these cytokines lead to specific differentiation signals. Although the tissue distribution of mitogens such as EGF, TGF alpha, amphiregulin, betacellulin, HB-EGF and cripto have been studied in detail, the physiological roles of these proteins have been difficult to determine. Clearly, these mitogens and the corresponding receptors are involved in the maintenance and progression of the tumorigenic state. The interactions between mitogenic, tumour suppressor and oncogenic systems are complex, but the tumorigenic effects of multiple lesions in intestinal carcinomas involve synergistic actions from lesions in these different systems. Together, the truncation of apc and activation of the ras oncogene are sufficient to induce colon tumorigenesis. If we are to improve cancer therapy, it is imperative that we discover the biological significance of these interactions, in particular the effects on cell division, movement and survival.     

Plasma membrane lipid order of leukemic and normal immature avian erythroid cells

Mammalian erythroblasts and their leukemic counterparts contain characteristic disordered regions of plasma membrane identified as putative membrane protein collection sites. In order to determine whether erythroid cells which do not enucleate contain homologous membrane domains, immature avian erythroid precursor cells and avian erythroleukemic cells were examined using merocyanine 540 (MC540), a fluorescent dye whose binding is sensitive to the packing of membrane lipids. Results were found to contrast with previous studies of the murine equivalents of these cells. In birds, normal erythroid precursors, including basophilic erythroblasts from the bone marrow and spleen of anemic animals, contained no detectable (less than 0.1%) cells which were stained by the dye. But cells from chicks infected with avian erythroblastosis virus (AEV) did stain. Considering the pattern of staining observed on AEV-erythroblasts relative to other leukemic and normal phenotypes, however, we conclude that neither normal nor leukemic avian erythroid cells contain a functional equivalent to the membrane protein collection sites found on their mammalian counterparts.     

Cell cycle regulation by growth factors and nutrients in normal and transformed cells

Summary SV3T3 cells, originally responsive to epidermal growth factor (EGF) and displaying density-dependent inhibition of growth, lose responsiveness to the growth factor after several passages and then proliferate without restriction, but continue to display EGF receptor sites at the cell surface. Proliferation of primary fetal rat hepatocytes is not stimulated by EGF, but cells bind it to an extent comparable to that of responsive 3T3 cells. Therefore presence of EGF receptors does not imply that cells are responsive to the growth factor. The relevance of some growth-factor-induced events for DNA synthesis initiation is dicussed. In various primary and secondary cell cultures, Ca⁺⁺-levels appear to be involved in controlling cell proliferation. In contrast, in 3T3-4a cells, levels of Ca⁺⁺ ions are not tightly coupled to DNA synthesis initiation; effects of growth factors are not mediated by extracellular Ca⁺⁺ ions, but cells have a Ca⁺⁺-sensitive restriction, point in G₁. In various cell types in primary or secondary culture or in 3T3-4a cells, polyamine, levels are not tightly coupled to induction of proliferation. Therefore growth-factor-induced ornithine decarboxylase is not an event essential for DNA synthesis initiation. Normal but not transformed cells have a spermidine/spermine-sensitive restriction point in G₁. Although rRNA synthesis appears to be necessary for induction of proliferation, preliminary data obtained by double-beam flow microfluorometry suggest that cellular RNA levels might not affect rate of entry into S phase and, furthermore, that 3T3-4a cells can enter S without accumulating RNA above levels present in quiescent cells. It appears that none of the events induced during the prereplicative phase that have been studied in 3T3 cells are essential for DNA synthesis initiation under normal culture conditions. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. This work was supported by Research Grants GM 20101, CA 15087, CA 14195, CA 12227 and CA 11176 from the USPHS, and Grant BC-30D from the American Cancer Society.     

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.     

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.     

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.     

Enucleation of normal and transformed cells

A quantitative analysis based on centrifugal force requirements for enucleation was developed to examine the response of a number of untransformed and transformed cell lines to cytochalasin mediated enucleation. Examination of the extent of cell enucleation as a function of centrifugal force resulted in a series of response curves demonstrating that enucleation g force requirements varied between Balb/c 3T3, Swiss 3T3, and Kirsten sarcoma virus transformed Balb/c 3T3 (3T3-K). A four times greater centrifugal force was required to reach 50% enucleation for transformed Balb/c 3T3-K when compared to Swiss 3T3. A qualitative correlation could be observed between ease of enucleation and the existence of a well-formed stress fiber network. A comparison of cytochalasin B and D suggested that cytochalasin D was far more effective in the enucleation of transformed cells. Experiments with 2-deoxyglucose and monensin provided evidence that decreasing cellular ATP levels, either directly or potentially by uncoupling ion transport from ATP generation, can decrease the efficiency of enucleation. It is suggested that the organization of the cytoskeleton is affected by the altered cellular ATP levels which can affect the centrifugal requirements of enucleation.     

Substrate flexibility regulates growth and apoptosis of normal but not transformed cells

One of the hallmarks of oncogenictransformation is anchorage-independent growth (27). Herewe demonstrate that responses to substrate rigidity play a major rolein distinguishing the growth behavior of normal cells from that oftransformed cells. We cultured normal or H-ras-transformedNIH 3T3 cells on flexible collagen-coated polyacrylamide substrateswith similar chemical properties but different rigidity. Compared withcells cultured on stiff substrates, nontransformed cells on flexiblesubstrates showed a decrease in the rate of DNA synthesis and anincrease in the rate of apoptosis. These responses on flexiblesubstrates are coupled to decreases in cell spreading area and tractionforces. In contrast, transformed cells maintained their growth andapoptotic characteristics regardless of substrate flexibility. Theresponses in cell spreading area and traction forces to substrateflexibility were similarly diminished. Our results suggest that normalcells are capable of probing substrate rigidity and that propermechanical feedback is required for regulating cell shape, cell growth,and survival. The loss of this response can explain the unregulated growth of transformed cells.

     

Ultrastructural localization of platelet-derived growth factor and related factors in normal and transformed cells

Visualization of platelet-derived growth factor (PDGF) and PDGF-like growth factors in cultured cells has been achieved by cryo-ultramicrotomy in combination with immunogold labeling. Immunogold staining of cryosections requires a mild chemical fixation in order to ensure preservation of antigenicity and ultrastructural details. Therefore the effect of several chemical fixatives on the antigenic properties of PDGF and PDGF-like growth factors was studied by indirect immunofluorescence using a polyclonal anti-PDGF antiserum. These studies demonstrated that formaldehyde has no effect on antigenicity, in contrast to glutaraldehyde or acrolein. For this reason formaldehyde was used as the only fixative for the visualization of PDGF in cryosections. PDGF was visualized in cryosections of normal human fibroblasts, preincubated with PDGF under various conditions. Preincubation at 4 degrees C with PDGF resulted in partial internalization of the growth factor. During subsequent warming of the cells to 37 degrees C PDGF was translocated to the nucleus. PDGF was also detected in the cytoplasm of tumor cells producing endogenous PDGF-like growth factors (neuroblastoma and simian sarcoma virus-transformed cells) but in these cases no significant amounts of these growth factors were present in the nucleus or at the extracellular surface of these cells. These results will be discussed in view of the intracellular routing of PDGF in normal responsive cells and of PDGF-like growth factors in factor-producing cells.     

Ecto-protein kinase activities in normal and transformed cells

The incubation of intact uninfected and Rous sarcoma virus (RSV)-transformed chicken cells (SR-RSV-A) with micromolar amounts of [gamma-32P]ATP under physiological conditions resulted in the radioactive phosphorylation of a variety of proteins. According to the experimental protocol the detectable phosphorylation was restricted to ATP utilization at the cell surface and was catalyzed by surface located protein kinase (PK). Serine- and to a lesser extent, threonine residues were phosphorylated. With respect to this enzyme the cells under investigation showed upon incubation with phosvitin the release of surface (phosvitin) kinase into the incubation medium. Based on immunochemical analysis and PK-assays using antisera from RSV-tumor bearing rabbits (TBR-serum) the pp60v-src with its associated tyrosine kinase activity was likewise detected in appreciable amounts at the outside of RSV-transformed chicken and mammalian cells. There was no cross reactivity of TBR-serum with phosvitin kinase. Phosvitin was not phosphorylated by the immunoprecipitated pp60v-src. Whereas phosphorylation catalyzed by pp60v-src was blocked with 10 to 20 microM diadenosine 5',5'-P1P4 tetraphosphate (Ap4A) the phosvitin phosphorylation was far less sensitive towards inhibition by Ap4A, similar to the cellular pp60c-src kinase activity in uninfected cells. The functional significance of the PK activities in uninfected and RSV-transformed cells observed at their surface or in cell-free form as well as the nature of their substrates remain to be established.     

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.     

Death-associated proliferation kinetic in normal and transformed cells

Pyruvate kinase M2 (PKM2) may occur in both a tetrameric and a dimeric form. When the majority of PKM2 molecules are in the highly active tetrameric conformation, glucose is primarily degraded to pyruvate and lactate with the regeneration of energy. A tumor suppressor protein, death-associated protein kinase (DAPK), interacts with PKM2 protein and stabilizes PKM2 in its active tetrameric form in normal proliferating cells. However, DAPK is widely inactivated in cancer cells, leading to the loss of the active conformation of PKM2. This may render PKM2 sensitive to cellular oxidants, switching the enzyme into its inactive dimeric form. Consequently, inhibition of PKM2 after oxidative stress contributes optimal tumor growth and allows cancer cells to withstand oxidative stress.     

Membrane lipid control of cytokinesis

In the final stage of cell division, cytokinesis constricts and then seals the plasma membrane between the two daughter cells. The constriction is powered by a contractile ring of actin filaments, and scission involves rearrangement of the lipid bilayer of the cell membrane. We have shown that the lipid phosphatidylethanolamine (PE), which normally resides in the internal leaflet of the bilayer, is exposed on the external leaflet of the cleavage furrow as a result of enhanced transbilayer movement of the phospholipids during cytokinesis. To investigate the role of PE in cytokinesis, we employed two different approaches: manipulation of cell surface PE by a PE-binding peptide and establishment of a mutant cell line specifically defective in PE biosynthesis. Both approaches provide evidence that surface exposure of PE is essential for disassembly of the contractile ring at the final stage of cytokinesis. Based on these findings, we proposed that the transbilayer redistribution of PE plays a critical role in mediating coordinated movements between the contractile ring and the plasma membrane that are required for the proper progression of cytokinesis.