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Study Objective Previous literature shows that women with endometriosis have higher rates of adverse pregnancy outcomes compared to the general population, including preterm birth, fetal growth restriction, and cesarean section. We aim to identify disease-specific risk factors and surgical treatments that impact obstetric outcomes. Design Retrospective cohort study. Setting Academic medical center. Patients or Participants Women who delivered at our institution from January 2002 to December 2018 with ICD-9/10 codes of endometriosis. Interventions We obtained demographic characteristics and pregnancy information from a prospectively-maintained dataset, and linked this to gynecologic data, including symptoms, infertility treatments, lesion types (superficial, deeply-infiltrative [DIE], endometrioma, adenomyosis), and prior surgeries for endometriosis—dichotomous variables for diagnostic laparoscopy, ablation/fulguration, ovarian cystectomy, superficial excision, and excision of DIE. Patients were stratified by gynecologic characteristics, and obstetric outcomes compared across groups. Measurements and Main Results Of 148 deliveries, 54 (36%) had superficial endometriosis, 14 (9.5%) DIE, 75 (51%) endometriomas, and 13 (8.8%) adenomyosis. Compared to other lesions, adenomyosis was associated with prior miscarriage (62% vs. 22%, p=0.004). Almost three-quarters (72%) of patients had undergone previous surgery, including 68% diagnostic laparoscopy, 22% ablation, 28% cystectomy, 8.8% superficial excision, and 4.1% DIE excision. 38% had previously been diagnosed with infertility, while 28% conceived through in vitro fertilization. Women with DIE more often delivered via cesarean section (71% vs. 40%, p=0.044). Compared to patients with zero or one prior surgery, the 25 patients with multiple prior surgeries (range 2 to >5) more frequently had small for gestational age infants (32% vs. 14%, p=0.015), which was independent of gestational age. There were no significant differences in obstetric outcomes (gestational age, delivery method or newborn weight) by type of endometriosis surgery. Conclusion More extensive surgical history, as evidenced by multiple prior endometriosis surgeries, was associated with having infants who were small for gestational age.

Determining the molecular identities of adult stem cells requires novel technologies for sensitive transcript detection in tissues. In mouse intestinal crypts, lineage-tracing studies suggested that different genes uniquely mark spatially distinct stem-cell populations, residing either at crypt bases or at position +4, but a detailed analysis of their spatial co-expression has not been feasible. Here we apply three-color single molecule fluorescence in-situ hybridization to study a comprehensive panel of intestinal stem-cell markers during homeostasis, aging and regeneration. We find that the expression of all markers overlap at crypt-base-cells. This co-expression includes Lgr5, Bmi1 and mTert, genes previously suggested to mark distinct stem cells. Strikingly, Dcamkl-1 tuft cells, distributed throughout the crypt axis, co-express Lgr5 and other stem cell markers that are otherwise confined to crypt bases. We also detect significant changes in the expression of some of the markers following irradiation, suggesting their potential role in the regeneration process. Our approach can enable the sensitive detection of putative stem cells in other tissues and in tumours, guiding complementary functional studies to evaluate their stem-cell properties.

Insulin-like growth factor binding proteins 1 and 3 are essentially known as regulators of IGF bioactivity. However, we previously showed that IGFBP-3 was able, in chick embryo fibroblast (CEF), to 100% inhibit DNA synthesis stimulated by calf serum, while the maximal inhibition found with IGFBP-1 was 60%, suggesting a difference between the two IGFBPs in their biological functions. Results of the present work agree with this assumption: (a) Recombinant human IGFBP-3, like rat IGFBP-3, was able to 100% inhibit DNA synthesis stimulation induced by human serum, while this stimulation was 75% decreased by IGFBP-1. However, the most striking difference was observed when the effects of the two IGFBPs were compared for stimulation induced by a serum growth factor (SGF) fraction depleted in IGFs. Stimulation induced by the SFG fraction was more significantly decreased (p0.001) by IGFBP-3 than by IGFBP-1. The mean percent inhibition +/- SEM was 67.1 +/- 2.5 in the presence of IGFBP-3 (200 ng/ml) and 29.3 +/- 2.7 and 34.2 +/- 4 in the presence of 200 and 400 ng/ml IGFBP-1 respectively. Inhibition by 200 ng/ml IGFBP-1 and inhibition by 6 ng/ml IGFBP-3 were additive. However, inhibition by IGFBP-3 and that by IGFBP-1 were no longer additive at high concentrations of IGFBP-3, which might thus replace IGFBP-1. (b) FGF stimulation of CEF was similarly inhibition (65% and 70%) by IGFBP-1 and IGFBP-3. (c) TGF beta stimulation of CEF was more strongly decreased by IGFBP-3 (90%) than by IGFB-1 (60%).(ABSTRACT TRUNCATED AT 250 WORDS)

Our results show that an insulin-like growth factor binding protein, IGFBP-3, purified from rat serum, is an inhibitor of chick embryo fibroblast (CEF) growth. It abolished DNA synthesis in CEF stimulated by IGF-I as well as by human serum. Rat IGFBP-3 and IDF45 (an inhibitory diffusible factor secreted by mouse cells) had the same activities, confirming that they have an intrinsic capacity to inhibit serum stimulation and may be considered as growth inhibitors. Our data show that inhibition by IGFBP-3 of serum stimulation was not simply the result of its inhibition of IGF present in the serum: 1) While anti-IGF-I IgG was able to completely inhibit stimulation induced by added IGF-I, it did not decrease stimulation induced by 1% human serum. Anti-IGF-II IgG inhibited the stimulation induced by added IGF-II, but only 25% decreased the stimulation induced by 0.7% serum. The percent inhibition was not significantly increased when the concentration of serum was decreased to 0.2%, which induced 140% stimulation of DNA synthesis; 2) stimulation by 0.2% serum was much more inhibited by IGFBP-3 than by IgG anti IGF-II; 3) after separation of IGF-I and IGF-II from serum by chromatography of acidified serum proteins on BioGel P150, the remaining serum proteins (with a molecular mass greater than 45 kDa) which were depleted in IGF-I and -II (verified by RIA determination) still stimulated DNA synthesis, and this stimulation was 80% inhibited by IGFBP-3.

From medium conditioned by 3T3 cells, we had previously purified an inhibitory factor of Mr 45 kDa which we termed IDF45 (inhibitory diffusible factor). The protein was able to 100% inhibit stimulation induced in CEF by 1% calf serum and to reversibly prevent cell growth. We then demonstrated that IDF45 was an IGF-binding protein. Our results suggested that IDF45 was a bifunctional molecule able to bind IGF and to inhibit DNA synthesis stimulated by this hormone, but also to inhibit stimulation of DNA synthesis induced by another growth factor in serum. Indeed, its N terminal amino acid sequence has great homology with that of IGFBP-3 and IDF45 is now proposed to be named IGFBP-3 (mouse IGF binding protein). Present results show that Ha-ras transfected 3T3 cells (EJ cells), like 3T3 cells, secrete a mIGFBP-3 molecule. In addition, transfected cells secrete a doublet of an IGF-binding protein (IGFBP-28) of Mr 28 kDa which is not secreted by untransformed 3T3 cells. IGFBP-28 has been purified and characterized in this work. Various results suggest that IGFBP-28 is not a degradation product of mIGFBP-3. Its N terminal amino acid sequence was different from that of mIGFBP-3. IGFBP-28 inhibited DNA synthesis stimulated by IGF-I, but much more IGFBP-28 protein than mIGFBP-3 was required to prevent this stimulation. In agreement with this result, IGFBP-28 has low affinity for IGF-I. In contrast, IGFBP-28 has high affinity for IGF-II. Like mIGFBP-3, IGFBP-28 was able to inhibit the stimulation induced by serum in CEF and to reversibly prevent growth, though with a specific activity lower than that of mIGFBP-3. It has also the capacity to inhibit stimulation of DNA synthesis induced by high molecular weight serum proteins depleted in IGF-I and II. In conclusion we have shown that transformation of 3T3 cells with Ha-ras induced the synthesis of a new IGF binding protein in medium conditioned by normal 3T3 cells. Our results suggest that IGFBP-28 like mIGFBP-3 is a bifunctional protein able to inhibit stimulation induced by IGF and by serum proteins different from IGFs.

We purified to homogeneity a growth inhibiting diffusible factor (IDF45) secreted by dense cultures of mouse 3T3 cells and which was able to inhibit 100% of DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF) (Blat et al., 1989a). We then demonstrated that this factor was an IGF-binding protein (Blat et al., 1989b). Indeed, its N-terminal amino acid sequence was homologous to that of rat IGFBP-3. Our present results show that basic fibroblast growth factor (bFGF) induced, respectively, a fivefold and threefold increase in DNA synthesis in mouse embryo fibroblasts (MEF) and CEF. IDF-45 inhibited the stimulation induced by bFGF by about 65%, while stimulation induced by insulin, PDGF, or EGF was only weakly or not at all inhibited by IDF45. When bFGF stimulation was determined in the presence of a high concentration of insulin in conditions which minimize the effect of endogenous IGF-I or -II, this stimulation was decreased by about 50% in the presence of IDF45. This result suggests that addition of bFGF stimulates IGF secretion, thereby resulting in partial loss of inhibition, by IDF45, of bFGF stimulation.

IDF45 (inhibitory diffusible factor) a mouse insulin-like growth factor binding protein (mlGFBP-3) has been shown to 100 percent inhibit DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF). Our previous results suggested that this large inhibition by IDF45 of serum stimulation was not just the result of its inhibitory activity toward IGF present in serum. The addition of Mn 2+ (10 −3 M) in the incubation medium enables us to show the presence of numerous binding sites per cells (about 60,000) of mlGFBP-3. However the dissociation constant (10 −8 M) indicated that this mouse IGFBP-3 bound to the membrane with low affinity. These findings lend new support to the assumption of the bifunctional property of IGFBP-3, which would have an effect outside the cell (binding of IGF in the medium) and another effect within cells or on the surface.

Mixed lineage kinase 3 (MLK3) is a MAP3K that activates the JNK-dependent MAPK pathways. Here, we show that MLK3 is required for cell migration in a manner independent of its role as a MAP3K or MLK3 kinase activity. Rather, MLK3 functions in a regulated way to limit levels of the activated GTPase Rho by binding to the Rho activator, p63RhoGEF/GEFT, which, in turn, prevents its activation by Galphaq. These findings demonstrate a scaffolding role for MLK3 in controlling the extent of Rho activation that modulates cell migration. Moreover, they suggest that MLK3 functions as a network hub that links a number of signaling pathways.

Factors inhibiting cell growth have been isolated from different cell types. However, little information is available concerning their mode of action. A novel growth inhibitory factor of 45 kDa (IDF45) was recently purified to homogeneity from medium conditioned by 3T3 cells. This molecule was able to inhibit DNA synthesis and the growth of chick embryo fibroblasts (CEF) in a reversible manner. By contrast, DNA synthesis stimulated by v-src expression in CEF was poorly inhibited by IDF45. In order to gain further insight into the IDF45 mode of action in normal and transformed CEF, we compared the effects of IDF45 on early stimulation of RNA synthesis induced in CEF by different mitogenic factors and by v-src gene expression. Stimulation, by serum, of RNA synthesis was inhibited by IDF45; however, inhibition increased when cells were preincubated with IDF45 before addition of serum and cell labeling for 2 h. IDF45 was also able to inhibit partially the stimulation of RNA synthesis induced by PMA and PDGF but was unable to inhibit stimulation of RNA synthesis induced by insulin and v-src expression. By contrast, stimulation of RNA synthesis induced by IGF-I was rapidly 100% inhibited by IDF45. The effect of IDF45 on DNA synthesis stimulated by the different mitogens was also determined and was correlated with the effect of IDF45 on RNA synthesis. These results suggest that the modes of action of IDF45 on stimulation of RNA synthesis by v-src and by insulin are similar. Our present results agree with others showing the bifunctional activity of IDF45 as an IGF-binding protein and as an inhibitory molecule in DNA stimulation induced by serum.

To pursue a systematic approach to the discovery of functional connections among diseases, genetic perturbation, and drug action, we have created the first installment of a reference collection of gene-expression profiles from cultured human cells treated with bioactive small molecules, together with pattern-matching software to mine these data. We demonstrate that this “Connectivity Map” resource can be used to find connections among small molecules sharing a mechanism of action, chemicals and physiological processes, and diseases and drugs. These results indicate the feasibility of the approach and suggest the value of a large-scale community Connectivity Map project.

Up until now the phenomenon of density dependent inhibition of growth has remained unexplained; one hypothesis suggests that autocrine growth inhibitory molecules are secreted in the medium of dense cultures and inhibit cell growth. From medium conditioned by mouse fibroblasts we purified a 45-kDa inhibitory factor which is an insulin-like growth factor binding protein (IGFBP-3). Based on different results we assumed that IGFBP-3 is a bifunctional molecule and has inhibitory function independent of its known function of binding IGF. In the present publication we attempted to verify whether IGFBP-3 is involved in DDI of growth. IGFBP-3 was secreted by mouse embryo fibroblasts (MEF). Its concentration in the medium increased with the cell density of the culture and was large at saturation density when DNA synthesis was minimum. Medium conditioned (CM) by dense culture was inhibitory compared to fresh medium and this inhibition disappeared when CM was preincubated with anti-IGFBP-3 IgG. Addition of FGFb to MEF dense cultures increased but transiently DNA synthesis which decreased as soon as 24 h after growth factor addition. By contrast accumulation of IGFBP-3 in the medium increased with time and was large at the time when DNA synthesis was minimum. Our results suggest that the rapid decrease of DNA synthesis in stimulated dense culture was the result of both depletion of the medium (particularly of FGFb) and the increase in concentration of inhibitory molecules like IGFBP-3. Addition of FGFb and preincubation of CM with anti-IGFBP-3 IgG were able to greatly reduce the inhibition.

miRNAs are post-transcriptional regulators of genes involved in such canonical signaling pathways as the TGF-β and EGF pathways, both of which are frequently altered in tumor cells. miRNA downregulation results in upregulation of its target genes and ultimately aberrant signaling of pathways involved in invasion and migration, for example. To better understand how alterations in miRNA expression could lead to metastasis, we used a lung adenocarcinoma mouse model in which a lentivirus expressing the Cre recombinase activates oncogenic K-Ras and deletes p53, giving rise to primary lung tumors (T) and metastases (M). We performed miRNA expression profiling on 30 T and M derived cell lines and identified three members of the miR-200 family - miR-200b, miR-200c, miR-429 - to be significantly downregulated in the M lines compared to their related T line. To explore the functional contribution of the miR-200s to metastasis, we designed a miRNA sponge to stably inhibit endogenous miR-200 levels in T cell lines. We found miR-200 depletion promoted wound healing and cell migration towards either serum or EGF. Transplantation of the sponge-expressing lines via tail vein led to in vivo formation of more invasive, metastatic tumors compared to controls. Previously, the miR-200 family has been shown to inhibit the TGF-β target gene and EMT mediator, Zeb1. We have identified another miR-200 target, Epidermal Growth Factor Receptor Substrate 8 (Eps8), which mediates actin based cell motility. In miR-200 low (M) cells, Eps8 is expressed three-fold higher than in corresponding miR-200 high (T) cells. Additionally, exogenous expression of Eps8 in T lines promotes wound healing and cell migration, relative to control cells. Thus, our findings implicate the miR-200s as a class of metastasis regulators that modulate expression of genes functionally relevant in tumor cell dissemination and provide new targets for therapeutic intervention of late stage lung adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B148.

Our results show that stimulation by serum of dense cultures of 3T3 cells rapidly induced increased synthesis of a growth inhibitor (mIGFBP-3) capable of binding IGF. mIGFBP-3 was secreted by stimulated cells immediately after its synthesis, and accumulated in the medium. Accumulation of mIGFBP-3 in the medium increased, as a function of growth factor (bFGF, PDGF, insulin) concentrations and time. bFGF was the best stimulatory factor for both DNA synthesis and accumulation of mIGFBP-3 in the first 24 h of incubation. DNA synthesis was arrested after 48 h of incubation with bFGF when accumulation of mIGFBP-3 was maximal. Since we showed that mIGFBP-3 is able to inhibit bFGF stimulation of DNA synthesis in mouse fibroblasts, it is possible that the accumulation of mIGFBP-3 induces a feedback regulation of cell growth.

We purified to homogeneity a growth inhibiting diffusible factor (IDF45) secreted by dense cultures of mouse 3T3 cells and which was able to inhibit 100% of DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF) (Blat et al., 1989a). We then demonstrated that this factor was an IGF-binding protein (Blat et al., 1989b). Indeed, its N-terminal amino acid sequence was homologous to that of rat IGFBP-3. Our present results show that basic fibroblast growth factor (bFGF) induced, respectively, a fivefold and threefold increase in DNA synthesis in mouse embryo fibroblasts (MEF) and CEF. IDF-45 inhibited the stimulation induced by bFGF by about 65%, while stimulation induced by insulin, PDGF, or EGF was only weakly or not at all inhibited by IDF45. When bFGF stimulation was determined in the presence of a high concentration of insulin in conditions which minimize the effect of endogenous IGF-I or -II, this stimulation was decreased by about 50% in the presence of IDF45. This result suggests that addition of bFGF stimulates IGF secretion, thereby resulting in partial loss of inhibition, by IDF45, of bFGF stimulation.

A novel cell growth inhibitor, IDF45 (inhibitory diffusible factor), was recently purified to apparent homogeneity. It is a bifunctional molecule: able to bind Insulin like growth factor (IGF) and to 100 % inhibit DNA synthesis stimulated by serum in fibroblasts. It was of interest to verify whether other members of the IGF-binding protein (IGFBP) family show the same bifunctional growth inhibitory properties. In this paper we show that purified IGFBP-1 derived from amniotic fluid is a cell growth inhibitor. In chick embryo fibroblasts, it inhibited DNA synthesis stimulated by serum. However the stimulation was maximally 60 % inhibited and half of the inhibition was observed with 100ng/ml IGFBP-1. So the specific activity of IGFBP-1 is lower than that of IDF45. IGFBP-1 also reversibly prevented the CEF growth. In the same cells IGFBP-1 inhibited DNA synthesis stimulated by IGF-I. We demonstrated that the same protein IGFBP-1 is able to inhibit DNA synthesis stimulated by serum and by IGF-I. The possibility that IGFBP-1 is a bifunctional molecule is discussed.

Deregulation of growth observed in malignant cell cultures has been assumed to be the result of increased secretion by these cells of autocrine growth factors, as well as the decreased sensitivity of these cells to inhibitory molecules which are diffused from normal or transformed cells. Our results show that PC-3 cells secreted into the medium, factors having stimulatory and inhibitory activities. We found an IGF-like molecule in medium conditioned by PC-3 cells. Its concentration was less than 1 ng/ml of conditioned medium. We demonstrated that PC-3 cells have receptors for IGF-I and are stimulated by this growth factor. However, the dose response curve shows that 1 ng/ml of IGF-I is not sufficient to indicate autocrine growth regulation by IGF of prostatic carcinoma cells. IGF-binding proteins of 90,000, 45,000, 34,000 and 28,000 molecular weight were also secreted by PC-3 cells. It is noteworthy that the secreted proteins which had the greatest inhibitory effect on chick embryo fibroblast growth also has the strongest IGF-binding activity. The probability that the IGF-binding protein secreted by PC-3 cells inhibited serum stimulation of DNA synthesis by preventing stimulation induced by IGF present in the serum is discussed. It is of interest that these IGF-binding proteins inhibited chick embryo fibroblast proliferation but did not inhibit PC-3 cells. This is in agreement with the assumption that IGF present in the medium is not an autocrine growth factor for these cells.

Low pressure chemical vapor deposition of polysilicon in a lamp heated rapid thermal processor (RTCVD) has been studied. Polysilicon films were deposited using SiH4 diluted in Ar. Structural characterization of the films was accomplished by transmissionelectron microscopy (TEM), scanning tunneling microscopy (STM), secondary ion mass spectroscopy (SIMS), auger electron spectroscopy (AES) and ultraviolet surface reflectance measurements. Smooth polysilicon films were obtained at deposition temperatures above 700ºC with rms roughness values better than 100 Å. Both p- and n- polysilicon gated MOS capacitors were fabricated using 80 - 200 Å thick gate oxides grown by dry oxidation in a conventional furnace. Polysilicon doping was achieved by ion-implantation and rapid thermal annealing (RTA). Our results show that the electrical properties ofthe capacitors fabricated using RTCVD polysilicon are comparable to those of conventional polysilicon. Dopant diffusion through the gate is a problem for both types of polysilicon and can lead to a degradation of the electrical properties.

A growth inhibitory protein named inhibitory diffusible factor 45 (IDF45) has been purified to homogeneity from medium conditioned by dense cultures of mouse 3T3 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of fast protein liquid chromatography-purified bioactive material showed a single band corresponding to a protein of 45 kDa. The molecule is a monomer. The pI of the molecule, as determined by isoelectric focusing, is about 6.5. The amino-terminal sequence of the protein was established as Ser-Ala-Gly-Ala-Val-Gly-Ala-Gly-Pro-Val-Val-Arg. The protein inhibits DNA synthesis in chicken embryo fibroblasts in a dose-dependent manner (ED50: 40 ng/ml, approximately 1 nM). The chemical properties of IDF45, i.e. molecular weight and amino-terminal sequence, clearly distinguish it from other known growth inhibitory proteins.

The synthesis and accumulation of RNA in Friend erythroleukemia (FL) cells induced to differentiate by treatment with the aminonucleoside of puromycin (AMS) or inhibited from differentiating by the addition of inosine to the medium were studied. When FL cells were grown in the presence of AMS, RNA synthesis was substantially inhibited. This effect could not be attributed solely to the inhibition of de novo purine synthesis because the biosynthesis of purine nucleotides from labeled inosine was much less depressed. The ratios of ATP to protein and of GTP to protein were slightly modified as compared to the untreated controls. However, the RNA/protein ratio was decreased. Thus, the RNA content of the cells was reduced 30-40%, but the protein content was not significantly affected. When the cells were treated with AMS together with inosine at a concentration that inhibits AMS-induced differentiation, the RNA/protein ratio was increased as compared with that found in cells treated with AMS alone and approached the level of the ratio in untreated control cells. Adenosine had a similar effect in overcoming the inhibition of RNA synthesis by AMS. Because the RNA/protein ratio of FL cells treated with dimethyl sulfoxide or sodium butyrate, two other potent inducers, was decreased by 44%, our results suggest that a correlation exists between the RNA content of the cells and the triggering of differentiation by inducers.

From medium conditioned by 3T3 cells, we had previously purified to apparent homogeneity a novel inhibitory diffusible factor of 45 kDa (IDF45), and then determined the amino-terminal sequence. IDF45 prevented reversibly the growth of chick embryo fibroblast (CEF). In these cells, DNA synthesis stimulated by 1% serum was 50% inhibited in the presence of 45 ng/ml (1 nM) IDF45. In the present article, we show that, in CEF, DNA synthesis stimulated by IGF-I was 100% inhibited in the presence of purified IDF45. Furthermore, the 45-kDa protein (IDF45) was, after Western blotting, able to bind IGF-I. The inhibitory effect of IDF45 upon serum stimulation did not seem to be the result of its inhibitory activity upon IGF-I stimulation, since stimulation by IGF-I and serum were additive. Moreover, it was possible to dissociate the two inhibitory effects: when added to v-src transformed CEF, IDF45 was able to 100% inhibit stimulation induced by IGF-I and was unable to significantly decrease stimulation induced by serum, as was previously observed. Taken together, our results strongly suggest that IDF45 has two distinct functions, one of which was to bind IGF-I and the other to inhibit serum stimulation. Indeed, it was impossible to separate the two functions when IDF45 was purified by cation exchange fast protein liquid chromatography, a method very different from reverse-phase fast protein liquid chromatography previously used for purification to apparent homogeneity of IDF45. On the other hand, if the IGF binding activity and inhibitory activity effect upon serum stimulation were carried by two different proteins, the presence of IGF-I (in conditions where most of the 45-kDa proteins were bound to IGF-I) should not have affected the activity of the molecule inhibiting serum stimulation. However, we observed the contrary: when IDF45 was bound to IGF-I, it lost its inhibitory effect upon stimulation induced by serum. This suggests that the two activities occurred on the same protein and that IDF45 is a bifunctional protein.

Phosphate uptake by monolayers of 3T3 cell decreases when the cultures enter the stationary phase, even when incubated in fresh medium containing 10% serum. However, SV 3T3 cultures retain a high rate of phosphate uptake when the cells reach saturation densities. We have observed that 3T3 cells grown to stationary phase in monolayers and then trypsinized and incubated in suspension, display an increase in phosphate uptake when the cell concentration is decreased from 10 6 cells/ml to 10 5 cells/ml. Where the cell concentration is further reduced from 10 5 cells/ml to 2.5 × 10 4 cells/ml there is no further increase in the rate of phosphate uptake. We observed, on the contrary, a small decrease. The “concentration effect” (the decrease of phosphate uptake when the cell concentration increases from 10 5 to 10 6 cells/ml) is larger when cells originate from a culture in stationary phase than when they originate from a culture in log phase. The “concentration effect” may be observed 10 min after cell incubation but is larger after a lag time of 40 min incubation. Differences in the “concentration effect” may be noted between 3T3 and SV 3T3 cells. In SV 3T3 cells no significant variations of phosphate uptake were observed when the cell concentration was changed. Thus, differences between phosphate uptake in 3T3 and SV 3T3 cells are large when cells are incubated at high concentrations or at high densities and small when they are incubated at low concentrations or at low densities. The “concentration effect” in 3T3 cells supports the assumption that interactions between cells cause the decrease of phosphate metabolism in dense culture. Diffusion of an inhibitor into the medium remains the more plausible explanation of the data.

Different data have suggested that the density-dependent inhibition of growth in normal 3T3 cells is due to the release of inhibitory factor(s) into the medium. The present results provide more evidence supporting this hypothesis. From conditioned medium of dense cultures of 3T3 cells, we fractionated a thermolabile inhibitory diffusible factor (I.D.F.) with a molecular weight of about 40.000 This factor decreased the [11C]-inosine incorporation into purine nucleotides and into nucleic acids of stimulated 3T3 cells. DNA synthesis, determined by [11C]-thymidine incorporation into nucleic acids, and protein synthesis (determined by the protein content of the cultures maintained in the presence or absence of I.D.F.), were also decreased. The possibility that I.D.F. is involved in density-dependent inhibition of growth is discussed.

The growth rate of normal cells multiplied in vitro decreases as the cell density of the culture increases. Previous results suggested that this density-dependent inhibition of growth in nontransformed cells was due to the diffusion of growth inhibitory substances in the medium of dense cultures. In this paper, we demonstrate that dense cultures of 3T3 cells secrete inhibitory and stimulatory factors. Macromolecules of conditioned medium were fractionated on Biogel P150 and the different fractions were tested on quiescent cultures of 3T3 cells stimulated or not to proliferate by addition of alpha globulin. When target cells were not stimulated to proliferate by addition of exocrine growth factors, we observed the inhibitory activity of a large molecular weight inhibitor (IDF45) and the stimulatory activity of autocrine growth factors (fraction about 35 and 10 K molecular weight), on the incorporation of 14C inosine into nucleotide pool and RNA. However, DNA synthesis was significantly stimulated with fraction 10 K only. This discrepancy between the stimulation of RNA and DNA synthesis may be explained by the presence, simultaneously, of inhibitory and stimulatory factors in fraction 35 and 10 K molecular weight. The presence of inhibitory factor was demonstrated when the fractions were tested on target cells stimulated to proliferate by alpha globulin addition and labeled with 14C thymidine. In these conditions, the stimulatory activity of autocrine growth factors was not observable, and only the inhibitory activity on DNA synthesis of fractions 35 and 10 K appeared. It is tempting to assume that the regulation of in vitro cell proliferation is determined by the balance between these antagonist stimulatory and inhibitory autocrine growth factors.

Recently we have characterized in phosphoproteins of plasma membranes from rat-liver cells three different sorts of phosphorylated groups: alkali-unstable phosphoserine and phosphothreonine, acid-unstable phosphohistidine and acylphosphate group unstable in hydroxylamine. It is shown here that the three groups were phosphorylated when the membranes were incubated with [γ- 32 P]ATP, during 30 sec. The degree of phosphorylation depended on conditions of incubation: 90% of the incorporated radioactivity was in acylphosphate phosphoproteins when the membranes were incubated at 0° with a low concentration of Mg 2+ . When the membranes were incubated at 25° with Mg 2+ (5 mM) only 43% of the incorporated radioactivity was hydroxylamine-unstable, 41% was acid-unstable and 16% alkali-unstable. Chase experiments have shown that the turnover of phosphate of the acyl-phosphate group was faster than that of phosphate of phosphohistidine. In contrast the phosphate of phosphoserine and phosphothreonine appeared to be relatively stable. Phosphorylation of acylphosphate and phosphohistidine phosphoproteins was especially Na + -, K + -dependent and was inhibited by ouabain as is (Na + K + )-dependent ATPase of the membranes. Our observations are consistent with the hypothesis that acylphosphate-bound phosphoproteins are related in the Na + -K + active transport across cell membranes. Our results suggest that ATP may not be the immediate precursor of phosphate of phosphoserine and phosphohistidine. In this case phosphorylation of these groups would be indirectly dependent on K + and ouabain.

Dilution of ascites tumor cells from 10 7 /ml to lower concentrations induced an enhancement of the amino acid uptake and protein synthesis. This phenomenonwill be referred to as “dilution effect”. It is shown that the enhancement of amino acid uptake is not closely dependent on he enhancement of protein synthesis. Puromycin inhibits 95% of the protein synthesis in cells incubated at the concentration 10 7 /ml and 78% in cells incubated at the concentration 10 6 /ml without any change in the amino acid uptake. Dilution of cells increases the active transport of amino acids which is related to the synthesis of ATP by oxidative phosphorylation: (a) The “dilution effect” is not observed at 0°, at which temmperature the active transport is negligible. (b) The “dilution effect” is markedly diminished, and occasionally abolished, by oligomycin and 2,4-dinitrophenol which are inhibitors of oxidative phosphorylation, and by glucose which decreases the respiration of ascites tumor cells. (c) Addition of glucose to oligomycin or to dinitrophenol induces the synthesis of glycolytic ATP which partially compensates for the loss of oxidative phosphorylation ATP, but does not proportionally restore the active transport of amino acids and the “dilution effect”.

Three different sorts of phosphoproteins have been characterized in plasma membranes from rat liver cells: alkali unstable phosphoproteins, containing phosphoseryl and phosphothreonyl groups: acid-unstablephosphoproteins containing phosphohistidine; and acyl phosphate-bound phosphoproteins. Alkali-unstable phosphoproteins from plasma membranes are more abundant than in any other structure (7 μg P/mg N protein as compared with 1.3 μg in microsomes). They are also more rapidly labelled. About 30 min after injecting 32P into animals, they attain a maximum specific radioactivity, which is 4 times greater than that of the total cell phosphoproteins. The maximum specific radioactivity is reached within the same time in acid-unstable phosphoproteins as well as in Pi and ATP, which suggests that a rapid exchange of 32P is occurring between these different compounds. In contrast, after labelling in vivo, acyl phosphate-bound phosphoproteins from plasma membranes are found to be 10–20 times less radioactive than alkali-unstable phosphoproteins, but they are very rapidly labelled in vitro when isolated membranes are incubated with [γ-32P]ATP. The radioactivity is K+ and Na+ dependent.

SummaryIntestinal crypts in mammals are comprised of long-lived stem cells and shorter-lived progenies. These two populations are maintained in specific proportions during adult life. Here, we investigate the design principles governing the dynamics of these proportions during crypt morphogenesis. Using optimal control theory, we show that a proliferation strategy known as a “bang-bang” control minimizes the time to obtain a mature crypt. This strategy consists of a surge of symmetric stem cell divisions, establishing the entire stem cell pool first, followed by a sharp transition to strictly asymmetric stem cell divisions, producing nonstem cells with a delay. We validate these predictions using lineage tracing and single-molecule fluorescence in situ hybridization of intestinal crypts in infant mice, uncovering small crypts that are entirely composed of Lgr5-labeled stem cells, which become a minority as crypts continue to grow. Our approach can be used to uncover similar design principles in other developmental systems.

We have previously shown [ 1 ] that some ribosomal proteins of rat liver were phosphorylated in vivo, on serine residues and that isolated ribosomes of rat liver could also be phosphorylated in vitro by ATP and a kinase from microsomal supernatant of rat liver. This phosphorylation was estimated by cyclic AMP at physiological doses (half-stimulation at 5 X 1 O-' M). It is well known that cyclic AMP is the cellular mediator of glucagon in rat liver [2] . Therefore we have studied the effects of glucagon on the in vivo phosphorylation of ribosomal proteins of rat liver. We have observed that 30 min after injection of glucagon and H, 32P0,, phosphorylation of ribosomal proteins was strongly increased. Autoradiography after electrophoresis of these proteins showed that phosphorylation of one of these proteins was stimulated 2 or 3 fold after injection of glucagon.

We here studied the protein kinase activity and in vitro phosphorylable sites of non-histone nuclear proteins, 0.4 M NaCl extracts (mostly chromosomal proteins) from chick embryo fibroblasts (CEF), infected or not with a Schmidt Ruppin strain subgroup A of Rous sarcoma virus (RSV). The infection and transformation of chick fibroblasts by RSV induced an increase in kinase activity and endogenous phosphorylation of non-histone chromosomal (NHC) proteins. The stimulation, by a change of medium, of the proliferation of dense cultures of normal chick fibroblasts also induced an increase in the kinase activity and endogenous phosphorylation of NHC proteins. However, two-dimensional gel electrophoresis of the 32 P-phosphorylated proteins showed that stimulation due to a change of medium and that due to the expression of transformation were very different. The stimulation by a change of medium increased to a greater or lesser extent the phosphorylation of the different NHC proteins, with no fundamental variations in the pattern of protein phosphorylation. In contrast, RSV infection induced significant changes in the pattern of protein phosphorylation. One of the most striking feature was the large increase of amount and phosphorylation of high molecular weight (HMW) proteins in particular of phosphoproteins having an evaluated molecular weight (MW) of 78 K and 82 K and pI>8.2. The percent of phosphotyrosine residues in NHC proteins was clearly increased when the proteins were extracted from transformed cells instead of normal cells. But the alkaline treatment of two-dimensional gel electrophoresis indicated that the 80 K phosphoproteins did not contain phosphotyrosine residues, and thus cannot be considered as substrates for pp60 src kinase.

The use of virus temperature-sensitive (ts) mutants for transformation allows us to determine the effect of transformation on different alterations in cell morphology and cell functions. We studied the kinase activity of nuclear non-histone proteins (NHP) extracted from chicken embryo fibroblasts (CEF), both infected and non-infected with a ts mutant of Rous sarcoma virus (Schmidt Ruppin strain, subgroup A, T class) and cultivated at the restrictive (41°C) and permissive (37 °C) temperature for transformation. The endogenous in vitro phosphorylation of NHP extracted from stationary ts-infected cultures increased by 300% when the cells were cultivated at 37 instead of 41°C. This increase is probably a consequence of the expression of transformation, since it was not observed when NHP were extracted from non-infected CEF maintained either at 41 or 37 °C. The increase in endogenous phosphorylation of NHP does not seem to be entirely due to a change in the density-dependent inhibition of cell metabolism in transformed cells, as an increase in protein phosphorylation was observed when the ts cultures were in both exponential growth phase and plateau phase. The urea-polyacrylamide gel electrophoresis of 32P-phosphorylated proteins showed that the increase in phosphorylation observed when growing ts cells were maintained at 37 instead of 41 °C was mostly due to the stimulation of one fraction (fraction d), as if the increase in phosphorylation of this fraction was linked to the expression of transformation. When the ts cultures were in stationary phase, the enhancement of the phosphorylation of proteins observed when cells were maintained at 37 instead of 41 °C was due to stimulation of the phosphorylation of all protein fractions, and particularly of fractions d and b. When phosvitin was used as a substrate, the kinase activity of NHP extracted from ts-infected cells was increased 300% when the cells were cultivated at 37 instead of 41 °C. On the other hand, the phosvitin kinase activity of NHP extracted from non-infected cells decreased when the cells were maintained at 37 instead of 41 °C. Thus, the transformation of cells induced an increase in the phosvitin kinase activity of nuclear NHP. When ts cells were cultivated at 41 °C until saturation density and then transferred to 37 °C for 3 h before extraction of proteins, a 37% increase in phosvitin kinase activity of NHP was observed, as compared with the activity of proteins extracted from cells left at 41 °C. Thus, the change in kinase activity is an early event in the process of transformation.

Density-dependent inhibition of growth has been assumed to be under the control of inhibitory molecules diffusing from dense cell cultures. Growth inhibitory factors have been fractionated or purified from medium conditioned by different cell types. In the present work, it was shown that IDF45 (inhibitory factor diffusing from 3T3 cells) decreased DNA synthesis in chick embryo fibroblasts (CEF) and was an inhibitor of CEF growth; this inhibition was reversible. Since similitudes between oncogene products and growth factors have been observed, it was of interest to compare the inhibitory effect of IDF45 upon the stimulation of DNA synthesis induced either by serum or by pp60-src. CEF infected by Ny68 virus (a mutant of Rous sarcoma virus ts for the expression of transformation) were density-inhibited at 41 degrees C, but were stimulated at this temperature by addition of 1% serum. This stimulation was 94% inhibited by IDF45. The same Ny68-infected cells could also be stimulated by transfer to 37 degrees C, the permissive temperature (in the absence of serum). The stimulation of DNA synthesis by src expression was poorly inhibited by IDF45. From our results, it appears that oncogene expression in CEF induces a loss in their sensitivity to IDF45. This would explain why transformed cells escape DDI of growth.

Similarities between the mode of action of growth factors and the oncogene product (pp 60 src protein) of Rous Sarcoma virus have been described. However, a major difference is that addition of growth factors does not induce a malignant transformation of cells. The present work proposes a hypothesis concerning this difference. Various data suggest that density-dependent inhibition (DDI) of growth in non-transformed cells is due to the diffusion of growth inhibitory molecules. Inhibitory factors of 45 K (IDF 45) and 12 K have been fractionated. We assume that the stimulation of DNA synthesis induced by growth factor addition to dense quiescent cultures of non-transformed cells leads to an increase in the activity of autocrine inhibitory molecules in such a manner that the growth factor stimulatory effect is only transient, and cells re-enter the Go phase. On the contrary, the stimulation of DNA synthesis by v-src transformation would not be counterbalanced by inhibitory diffusing factors and cells would not enter Go phase. We present preliminary results which support this assumption. Dense quiescent cultures of chick embryo fibroblasts infected by Ny 68 virus (ts mutant for transformation of Rous Sarcoma virus) were stimulated to proliferate either by addition of growth factors in cultures maintained at 41 degrees C or by expression of transformation (by the cell transfer from 41 to 37 degrees C, the permissive temperature for expression of transformation). Stimulation of DNA synthesis by growth factors was totally inhibited by the inhibitory diffusing factors of 45 K (IDF45) whereas the stimulation of DNA synthesis produced by transformation was reproducibly not decreased by IDF45.

An inhibitory diffusible factor of 45 kDa (IDF45) was isolated from medium conditioned by dense cultures of 3T3 cells. The procedure involved Bio-Gel PI 50 chromatography and 2 reverse-phase FPLC. After the final step of purification, 60 ng ml of IDF45 inhibited 50% of α-globulin-stimulated DNA synthesis. It was shown that IDF45 acted in the G1 phase of the cell cycle. When added for 8 h in the G1 phase of the cell cycle, it was able to inhibit DNA synthesis in the S phase which followed this G1 phase. Furthermore, IDF45 inhibited the early stimulation of RNA synthesis induced by α-globulin.

We have compared the in vivo phosphorylation of the non-histone chromosomal (NHC) proteins in relation to nucleic acid synthesis, in cells stimulated to proliferate and in resting cells. When BHK21C13 cells in saturation density are stimulated to proliferate by a change of medium, a synchronized DNA synthesis occurs with a maximum at about 16 h. Early after stimulation the NHC protein phosphorylation increases and reaches a maximum in late G 1 before the onset of DNA synthesis. It remains high during early S and decreases in late S. The phosphorylation of total cellular proteins, on the contrary, is increased only at the time of maximum DNA synthesis and always remains lower than that of NHC proteins. Early after stimulation and through the G 1 phase an increase of total RNA synthesis is also observed, however the time of maximum RNA synthesis does not coincide with the maximum of NHC protein phosphorylation. The involvement of NHC protein phosphorylation in the regulation of some messenger RNA (mRNA) synthesis is discussed.