Puromycin-induced necrosis of crypt cells of the small intestine of mouse.

Chemical and radioautographic analysis of the small intestine of mice injected intraperitoneally with puromycin revealed an immediate decrease of precursor incorporation into DNA and protein and a delayed decrease of precursor incorporation into RNA. In addition to this decrease of precursor incorporation, damage to the crypt cells, but not to the cells of the villus of the small intestine, was observed. Further examination of other dividing cells (spleen) and nondividing cells (liver and heart) of these mice showed again that only cells of actively dividing tissues were damaged. The metabolic inhibitors actinomycin D, cytosine arabinoside, actidione, and puromycin aminonucleoside were used in an attempt to clarify the mechanism of cell damage by puromycin. The results showed that there was no clear correlation between cell necrosis and the pattern of inhibition of synthesis of DNA, RNA, or protein.

Changes in chromatin template activity and their relationship to DNA synthesis in mouse parotid glands stimulated by isoproterenol.

Chromatin template activity of mouse parotid glands increases after a single injection of isoproterenol (IPR), a procedure that causes, after a lag period of 20 hr, a marked stimulation of DNA synthesis and cell division in salivary glands of rodents. The increase in chromatin template activity occurs as early as 1 hr and peaks between 6 and 10 hr after IPR, paralleling previously reported changes in the incorporation of uridine-(3)H into total cellular RNA of mouse parotids. Template activity was measured in vitro in a system in which parotid gland chromatin was incubated with an exogenous RNA polymerase isolated from Escherichia coli. Similar results were obtained when template activity of parotid gland chromatin was assayed using an homologous RNA polymerase from mouse liver. Chromatin template activity in mouse parotids was also studied after the administration of drugs capable of inducing in salivary glands both DNA synthesis and secretion or secretion alone. The results indicate that the increased chromatin template activity occurring 6 hr after IPR is related to the subsequent onset of DNA synthesis. Furthermore, the increased chromatin template activity caused by IPR is inhibited by the previous administration of puromycin, an inhibitor of IPR-stimulated DNA synthesis.

Control of hsp70 RNA levels in human lymphocytes.

The expression of a hsp70 gene in human cells has previously been shown to be related to the growth state of the cells. As an alternative to in vitro synchronization procedures, we have measured steady-state levels of the RNA for a heat-shock protein 70 (hsp70) in human peripheral blood mononuclear cells (PBMC) that are naturally quiescent in a G0 state. The probe used recognized, on RNA blots, one single band. The levels of this hsp70 RNA are elevated in circulating PBMC and decrease when the cells are incubated with serum, or phytohemagglutinin, or simply when they are incubated in culture medium. The levels of hsp70 RNA decrease within 30 min after in vitro culture, and are accompanied by an increase in the levels of c-fos RNA. These findings, together with other recent reports in the literature, suggest a possible role of the hsp70 proteins in the regulation of cell growth.

Polyinosinic acid--polycytidylic acid: inhibition of DNA synthesis stimulated by isoproterenol.

Polyinosinic acid * polycytidylic acid (poly I * poly C) inhibits isoproterenol-stimulated DNA synthesis in salivary glands of mice. A single intraperitoneal injection of 250 micrograms of poly I * poly C inhibits the stimulation of DNA synthesis when given 10 minutes before isoproterenol or at any time during the 20-hour lag period between the injection of isoproterenol and the subsequent DNA synthesis. The inhibition caused by poly I * poly C is not due to a generalized toxic action but seems to be exerted through a relatively selective mechanism. Polyuridylic acid and diethylaminoethyl dextran are less effective in inhibiting isoproterenol-stimulated DNA synthesis.

Uridylate kinase activity: effect of isoproterenol.

Isoproterenol stimulates cell proliferation in mouse salivary glands. Prior to the stimulation of DNA synthesis, (3)H-uridine incorporation into RNA is decreased. This decreased incorporation results from a depression of uridylate kinase activity.

DNA structure in sheared and unsheared chromatin.

Shearing chromatin, by either sonication or vortex homogenization, introduces significant structural artifacts. These may be detected by the anomalously large increase in the number of ethidium bromide binding sites and the large alteration of the circular dichroism spectra of chromatin. Structural alterations are also suggested by the disappearance of differential light scattering after shearing.

Stimulated DNA synthesis in frog nuclei by cytoplasmic extracts of temperature-sensitive mammalian cells.

Cytoplasmic extracts of proliferating cells stimulate DNA synthesis in isolated nuclei of Xenopus laevis liver. When tested by the same assay, cytoplasmic extracts of resting cells are completely inactive. When cytoplasmic extracts are prepared from cell cycle-specific temperature-sensitive mutants arrestd in the G1 phase of the cell cycle by the nonpermissive temperature, they also fail to stimulate DNA synthesis in frog nuclei. The results indicate that, to stimulate DNA synthesis in isolated frog nuclei, essentially all information of G1 cells must be present.

Inhibition of cellular proliferation by antisense oligodeoxynucleotides to PCNA cyclin.

The proliferating cell nuclear antigen (PCNA or cyclin) is a nuclear protein recently identified as a cofactor of DNA polymerase delta. When exponentially growing Balb/c3T3 cells are exposed to antisense oligodeoxynucleotides to PCNA, both DNA synthesis and mitosis are completely suppressed. A corresponding sense oligodeoxynucleotide has no inhibitory effects. These experiments indicate that PCNA (cyclin) is important in cellular DNA synthesis and in cell cycle progression.

Microinjected c-myc as a competence factor.

While a number of oncogenes are expressed in a cell cycle-dependent manner, their role in the control of cell proliferation can only be established by a direct functional assay. The c-myc protein, upon microinjection into nuclei of quiescent Swiss 3T3 cells, cooperated with platelet-poor plasma in the stimulation of cellular DNA synthesis. This suggests that c-myc protein, like platelet-derived growth factor (PDGF), may act as a competence factor in the cell cycle to promote the progression of cells to S phase. The presence in the medium of an antibody against PDGF abolished DNA synthesis induced by microinjected PDGF; however, the microinjected c-myc protein stimulated DNA synthesis even when its own antibody was present in the medium. The c-myc protein may act as an intracellular competence factor, while PDGF expresses its biological activity only from outside the cells.

Transformation by the simian virus 40 T antigen is regulated by IGF-I receptor and IRS-1 signaling.

Previous work has shown that the Simian Virus 40 T antigen (T antigen) cannot transform mouse embryo fibroblasts (MEFs) that do not express the type 1 insulin-like growth factor receptor (IGF-IR). We have now investigated the mechanism(s) by which the transforming activity of T antigen is affected by IGF-IR signaling. We demonstrate that transformation by T antigen of MEFs and several other cell lines requires an insulin receptor substrate-1 (IRS-1) phosphorylated on tyrosines. If IRS-1 is not expressed, or is serine phosphorylated or otherwise inactive, T antigen fails to transform cells in culture. For instance, while T antigen cannot transform 32D myeloid cells (that do not express IRS-1), its transforming activity is restored by the expression of a wild-type IRS-1, but not of an IRS-1 mutated at the PI3K binding sites. The importance of IRS-1 activation of PI3K in T-antigen transformation is supported by the finding that a constitutively activated p110 subunit of PI3K, a target of IRS-1, overcomes the inability of T antigen to transform MEFs with a serine phosphorylated IRS-1. Taken together, these results indicate that the IRS-1/PI3K signaling is one of the mechanisms regulating transformation by the SV40 T antigen. We propose that the requirement for a tyrosyl-phosphorylated IRS-1 provides a mechanism to explain the failure of T antigen to transform MEFs with deleted IGF-IR genes.

Overexpression of insulin-like growth factor-1 in mice protects from myocyte death after infarction, attenuating ventricular dilation, wall stress, and cardiac hypertrophy.

To determine whether IGF-1 opposes the stimulation of myocyte death in the surviving myocardium after infarction, transgenic mice overexpressing human IGF-1B in myocytes (FVB.Igf+/-) and wild-type littermates at 1.5 and 2.5 mo of age were subjected to coronary ligation and killed 7 d later. Myocardial infarction involved an average 50% of the left ventricle, and produced cardiac failure. In the region proximate to infarction, myocyte apoptosis increased 4. 2-fold and 2.1-fold in nontransgenics at 1.5 and 2.5 mo, respectively. Corresponding increases in myocyte necrosis were 1. 8-fold and 1.6-fold. In contrast, apoptotic and necrotic myocyte death did not increase in FVB.Igf+/- mice at either age after infarction. In 2.5-mo-old infarcted nontransgenics, functional impairment was associated with a 29% decrease in wall thickness, 43% increase in chamber diameter, and a 131% expansion in chamber volume. Conversely, the changes in wall thickness, chamber diameter, and cavitary volume were 41, 58, and 48% smaller in infarcted FVB.Igf+/- than in nontransgenics. The differential response to infarction of FVB.Igf+/- mice resulted in an attenuated increase in diastolic wall stress, cardiac weight, and left and right ventricular weight-to-body wt ratios. In conclusion, constitutive overexpression of IGF-1 prevented activation of cell death in the viable myocardium after infarction, limiting ventricular dilation, myocardial loading, and cardiac hypertrophy.

Functional analysis and growth factor regulation of the human vimentin promoter.

Vimentin is a growth-regulated gene whose mRNA levels increase severalfold after stimulation of quiescent cells. We have isolated and sequenced a genomic fragment of human DNA containing the vimentin 5'-flanking sequence and untranslated region. S1 nuclease analysis was used to determine the transcription initiation site. Deletion mutants of the promoter region were constructed, linked to a chloramphenicol acetyltransferase gene, and analyzed for transient expression by transfection into BALB/c 3T3 cells. These experiments revealed the presence in the human vimentin promoter region of a negative-regulatory element, flanked by positive elements. The most 5' of the positive elements is able to overcome the effects of the negative element. Analysis of these deletion constructs in stable cell lines confirmed the results of the transient assays. Using these stable cell lines, we can also demonstrate that the vimentin promoter region can confer platelet-derived growth factor inducibility to a linked chloramphenicol acetyltransferase gene and that the sequences required for this inducibility reside between positions -241 and +73.

Induction of cellular deoxyribonucleic acid synthesis in butyrate-treated cells by simian virus 40 deoxyribonucleic acid.

Sodium butyrate (3 mM) inhibited the entry into the S phase of quiescent 3T3 cells stimulated by serum, but had no effect on the accumulation of cellular ribonucleic acid. Simian virus 40 infection or manual microinjection of cloned fragments from the simian virus 40 A gene caused quiescent 3T3 cells to enter the S phase even in the presence of butyrate. NGI cells, a line of 3T3 cells transformed by simian virus 40, grew vigorously in 3 mM butyrate. Homokaryons were formed between G1 and S-phase 3T3 cells, Butyrate inhibited the induction of deoxyribonucleic acid synthesis that usually occurs in B1 nuclei when G1 cells are fused with S-phase cells. However, when G1 3T3 cells were fused with exponentially growing NGI cells, the 3T3 nuclei were induced to enter deoxyribonucleic acid synthesis. In tsAF8 cells, a ribonucleic acid polymerase II mutant that stops in the G1 phase of the cell cycle, no temporal sequence was demonstrated between the butyrate block and the temperature-sensitive block. These results confirm previous reports that certain virally coded proteins can induce cell deoxyribonucleic acid synthesis in the absence of cellular functions that are required by serum-stimulated cells. Our interpretation of these data is that butyrate inhibited cell growth by inhibiting the expression of genes required for the G0 leads to G1 leads to S transition and that the product of the simian virus 40 A gene overrode this inhibition by providing all of the necessary functions for the entry into the S phase.

Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies.

Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb 122 inhibits the transition of Swiss 3T3 cells from G0 to S phase, but has no effect on the progression of these cells from mitosis to the S phase.

Activation of the ribosomal DNA promoter in cells exposed to insulinlike growth factor I.

We constructed a stable cell line, 3T3A5, which carried a chimeric gene in which the simian virus 40 T-antigen-coding gene was under the control of the mouse ribosomal DNA promoter. These cells expressed T antigen when they were growing exponentially in 10% fetal calf serum, but they all became T negative when incubated for 5 days in low-concentration serum. The readdition of serum or platelet-poor plasma again induced the expression of T antigen, which was accompanied by an increase in steady-state levels of the corresponding RNA. Among the various growth factors tested for their ability to induce T-antigen expression in 3T3A5 cells, only insulinlike growth factor I (IGF-I) could induce T antigen at physiological concentrations. The effect of IGF-I or platelet-poor plasma was abolished by an antibody to IGF-I. Other growth factors, like insulin and epidermal growth factor, could induce the expression of T antigen in 3T3A5 cells, but only at concentrations far above the physiological range. Other growth factors were totally ineffective. These results indicate that exposure of cells to IGF-I can activate transcription from the ribosomal DNA promoter.

Regulation of Id gene expression by type I insulin-like growth factor: roles of Stat3 and the tyrosine 950 residue of the receptor.

Id proteins are known to play important roles in the proliferation and differentiation of many cell types. The type 1 insulin-like growth factor receptor (IGF-IR), activated by its ligand, induces the differentiation of 32D IGF-IR cells, a murine hematopoietic cell line, expressing a human IGF-IR. Expression in 32D IGF-IR cells of a dominant negative mutant of Stat3 (DNStat3) inhibits IGF-I-mediated differentiation. DNStat3 causes a dramatic increase in Id2 gene expression. This increase, however, is IGF-I dependent and is abrogated by a mutation at tyrosine 950 of the IGF-IR. These results indicate that in 32D cells, the IGF-IR regulates the expression of the Id2 gene and that this regulation is modulated by both positive and negative signals. Our results also suggest that in this model, Id2 proteins influence the differentiation program of cells but are not sufficient for the full stimulation of their proliferation program.

Insulin-like growth factor I receptor signaling in transformation by src oncogenes.

R- cells, a line of mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor I (IGF-I) receptor genes, are refractory to transformation by several viral and cellular oncogenes. Using colony formation in soft agar as a measure of full transformation, we report here that R- cells can be transformed by v-src, although they still cannot be transformed by the activated c-src527 (mutation at tyrosine 527 to phenylalanine), which readily transforms mouse embryo cells with a wild-type number of IGF-I receptors (W cells). Although v-src is a more potent inducer of tyrosine phosphorylation than c-src527, the extent of phosphorylation of either insulin receptor substrate 1 or Shc, two of the major substrates of the IGF-I receptor, does not seem sufficiently different to explain the qualitative difference in soft agar growth. v-src, however, is considerably more efficient than c-src527 in its ability to tyrosyl phosphorylate, in R- cells, the focal adhesion kinase, Stat1, and p130cas. These results indicate that v-src, but not c-src527, can bypass the requirement for a functional IGF-I receptor in the full transformation of mouse embryo fibroblasts and suggest that qualitative and quantitative differences between the two oncogenes can be used to identify some of the signals relevant to the mechanism(s) of transformation.

Adenovirus type 2 activates cell cycle-dependent genes that are a subset of those activated by serum.

We have studied a panel of 10 genes and cDNA sequences that are expressed in a cell cycle-dependent manner in different types of cells from different species and that are inducible by different mitogens. These include five sequences (c-myc, 4F1, 2F1, 2A9, and KC-1) that are preferentially expressed in the early part of the G1 phase, three genes (ornithine decarboxylase, p53, and c-rasHa) preferentially expressed in middle or late G1, and two genes (thymidine kinase and histone H3) preferentially expressed in the S phase of the cell cycle. We have studied the expression of these genes in nonpermissive (tsAF8) and semipermissive (Swiss 3T3) cells infected with adenovirus type 2. Under the conditions of these experiments, adenovirus type 2 infection stimulates cellular DNA synthesis in both tsAF8 and 3T3 cells. However, four of the five early G1 genes (c-myc, 4F1, KC-1, and 2A9) and one of the late G1 genes (c-ras) are not induced by adenovirus infection, although they are strongly induced by serum. The other sequences (2F1, ornithine decarboxylase, p53, thymidine kinase, and histone H3) are activated by both adenovirus and serum. We conclude that the cell cycle-dependent genes activated by adenovirus 2 are a subset of the cell cycle-dependent genes activated by serum. The data suggest that the mechanisms by which serum and adenovirus induce cellular DNA synthesis are not identical.

Role of the promoter in the regulation of the thymidine kinase gene.

To identify the regulatory elements of the human thymidine kinase (TK) gene, we have established stable cell lines carrying different chimeric constructs of the TK gene. Our results can be summarized as follows. (i) When the TK coding sequence is under the control of the calcyclin promoter (a promoter that is activated when G0 cells are stimulated by growth factors), TK mRNA levels are higher in G1-arrested cells than in proliferating cells; (ii) when the TK coding sequence is under the control of the promoter of heat shock protein HSP70, steady-state levels of TK mRNA are highest after heat shock, regardless of the position of the cells in the cell cycle; (iii) the bacterial CAT gene under the control of the human TK promoter is maximally expressed in the S phase; (iv) the TK cDNA driven by the simian virus 40 promoter is also maximally expressed in the S phase; and (v) TK enzyme activity is always at a maximum in the S phase, even when the levels of TK mRNA are highest in nonproliferating cells. We conclude that although the TK coding sequence may also play some role, the TK promoter has an important role in the cell cycle regulation of TK mRNA levels.

Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells.