Human alpha-fetoprotein purified from amniotic fluid enhances growth factor-mediated cell proliferation in vitro.

Using a primary monolayer culture of porcine granulosa cells (pGC) as an in vitro cell proliferation assay, we have examined the growth-promoting activity of alpha-fetoprotein (AFP) purified from term cord blood and midtrimester amniotic fluid. Increasing concentrations (2.5-20%) of crude human cord blood (CB) increased pGC proliferation, while identical concentrations of crude amniotic fluid (AF) were ineffective. When the cell system was maximally stimulated, AF dose dependently decreased cell proliferation. AFP purified from AF and CB (1.25-5.0 micrograms/ml) was not mitogenic alone, but, in the presence of epidermal growth factor (EGF) + insulin-like growth factor (IGF-I) (10 ng/ml each), AFP dose dependently increased cell proliferation to nearly double that of EGF + IGF-I alone. The response of pGC to the proliferative effects of AF-AFP and CB-AFP were identical at each dose of AFP tested. These results indicate that although crude, pooled midtrimester AF does not display the mitogenic activity seen in cord blood, AFP purified from pooled AF significantly synergizes with growth factors to increase cell proliferation markedly.     

To best measure cell proliferation in samples from the intestine

Arrangement of the intestinal cell lining, as it is, into distinct anatomically defined zones where proliferation is confined to the crypts, makes it an ideal tissue to study growth control mechanisms. While many methods have been used to quantify cell proliferation in the gut, several of them have severe limitations and others (although potentially better) have been misused and misinterpreted. Here, correct use and interpretation of labelling studies will be described as will a well established alternative method that provides equivalent results for one-sixth of the effort.     

T cell growth factors from adult T cell leukemia virus-transformed cell lines

Some characteristics of T cell growth factors derived from adult T cell leukemia virus (ATLV)-transformed cell lines, MT 1 and MT 2 were analyzed. MT 1 cells release significant interleukin 2 (IL 2) activity into the culture medium, which showed the same elution pattern of gel filtration and isoelectric focusing of IL 2 from lectin-stimulated normal human lymphocytes. This activity was also detected in the cell extract of MT 1. In contrast, MT 2 cell line did not produce IL 2 activity, but non-IL 2 type growth factor was observed. The significance of these factors from MT cell lines is discussed from the viewpoint of 'autokine' in ATLV-transformed cells.     

Regulation of cell proliferation by epidermal growth factor

Epidermal Growth Factor (EGF) is a 6045 dalton polypeptide which stimulates the proliferation of various cell types in vitro and in vivo. EGF binds to diffusely distributed membrane receptors which rapidly cluster primarily on coated pits areas on the plasma membrane. Subsequently, the EGF-receptor complexes are endocytosed and degraded by lysosomal enzymes. The lateral diffusion coefficient (D) of EGF-receptor complexes on cultured cells increases gradually from D = 2.8 X 10(-10) cm2/sec at 5 degrees C to 8.5 X 10(-10) cm2/sec at 37 degrees C. In the same range of temperature the rotational correlation times change from 25 to 50 microseconds to approximately 350 microseconds. Hence, at 4 degrees C, the occupied EGF receptors translate and rotate rapidly in the plane of the membrane. At 37 degrees C, EGF receptors form microclusters composed of 10 to 50 molecules. Moreover, it is concluded that both at 4 degrees C and 37 degrees C lateral diffusion of the occupied receptors is not the rate determining step for either receptor clustering or internalization. EGF receptor is a 150,000 to 170,000 dalton glycoprotein. The receptor is in close proximity to an EGF-sensitive, cAMP-independent, tyrosine-specific protein kinase which also phosphorylates the receptor molecules itself. The EGF sensitive kinase is similar to the kinase activity which is associated with certain RNA tumor viruses. The fact that the non-mitogenic cyanogen-bromide cleaved EGF is as potent as native EGF in stimulating phosphorylation suggests that EGF-induced, protein phosphorylation is a necessary but insufficient signal for the induction of DNA synthesis by EGF. EGF receptor serves also as the binding site for Transforming Growth Factors (TGF) which compete with EGF and induce anchorage-independent growth of normal cells in soft agar. Tumor promoters such as phorbol ester effect the binding of EGF to its membrane receptors and its ability to stimulate DNA synthesis. EGF itself has also some tumor promoting activity. Hence, the membrane receptor for EGF seems to participate in the regulation of normal and neoplastic growth. Monoclonal antibodies against EGF receptor (IgM) induce various early and delayed effects of EGF, while their monovalent Fab' fragments are devoid of biological activity. These observations support the notions that EGF receptor rather than EGF itself is the active moiety and that the role of the hormone is to perturb the receptor in the appropriate way, probably by inducing the microaggregation of EGF receptors.     

Canstatin-N fragment inhibits in vitro endothelial cell proliferation and suppresses in vivo tumor growth

Type IV collagen is one of the components of vascular basement involved in regulation of angiogenesis. Canstatin, the non-collagenous 1 (NC1) domain of alpha2 chain of type IV collagen, was identified as an inhibitor of angiogenesis and tumor growth by Kamphaus et al. Our previous studies showed that canstatin-N, the N-terminal 1-89 amino acid fragment of canstatin, inhibited the neovascularization in a dose-dependent manner as tested by CAM assay. In the present study, we demonstrate that canstatin-N produced in Escherichia coli specifically inhibited in vitro the proliferation of human umbilical vein endothelial cells (ECV304) and significantly induced apoptosis. The apoptosis-inducing activity of canstatin-N was much stronger than that of canstatin, indicating that the apoptosis-inducing activity of canstatin is likely located within its N-terminal 1-89 amino acid fragment. Canstatin-N also suppressed in vivo growth of B(16) murine melanoma in BALB/c mice at a dosage of 10mg/kg/day. These results suggest that canstatin-N is a useful candidate molecule for inhibition of tumor growth.     

The stimulation of normal human melanocyte proliferation in vitro by melanocyte growth factor from bovine brain

Cell culture conditions for the selective growth and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of 2% fetal bovine serum, the human melanocyte cell culture environment contains the following growth factor supplements: epidermal growth factor (10 ng/ml), triiodothyronine (10(-9) M), hydrocortisone, (5 X 10(-5) M), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), 7S nerve growth factor (100 ng/ml) cholera toxin (10(-10) M), and bovine brain extract (150 micrograms/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal keratinocytes and melanocytes for attachment to fibronectin, while the growth of the human melanocyte has been attributed to the mitogenic activity of the growth factor-supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]-Dopa incorporation. The growth factor-supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte growth factor is described as a heat and alkaline-labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the growth of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co-cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilities of human melanoma.     

Age-related and carcinogen-induced alterations of the extracellular growth factor requirements for cell proliferation in vitro

Cells from thigh muscles of fetal rats proliferate readily in vitro in medium containing homologous adult rat "plasma". As the donor animals mature, these cells become unable to make DNA and proliferate in "plasma" medium, but retain an ability to proliferate in medium containing fetal bovine serum (FBS). This age-related loss of the ability to proliferate in "plasma"-medium is due to an increasing need by the cells for an exogenous prereplicative promoter which is found in FBS and a crude preparation of bovine luteinizing hormone. Adult cells (and possibly fetal cells) also require a "cycle-completion" factor which is found in FBS and adult rat "plasma". The requirements for such external proliferative promoters is completely eliminated by neoplastic transformation in vivo, and neoplastic adult cells isolated from a nickel sulfide-induced mixed rhabdomyo-fibro-sarcoma can make DNA and proliferate in vitro in the complete absence of exogenous growth factors.     

Effect of endothelial cell growth supplement and fibroblastic growth factor on early mouse embryo development in vitro

This study was conducted to examine the effect of endothelial cell growth supplement (ECGS) and fibroblastic growth factor (FGF) on early mammalian embryo development in vitro. Two hundred mouse blastocysts were placed randomly in culture wells containing one of five treatments: 1) Ham's F-10, 2) Ham's F-10 + 10 mug ECGS, 3) Ham's F-10 + 10 ng ECGS, 4) Ham's F-10 + 100 ng FGF and 5) Ham's F-10 + 10 ng FGF. In all cases, media were supplemented with 10% (v/v) normal steer serum. Embryos were cultured at 37 C with an atmosphere of 5% O(2), 5% CO(2) and 90% N and development was recorded at 12 h intervals for the duration of culture. The percentage of embryos that developed to expanded blastocyst (94.8), hatching blastocyst (74.4) and hatched blastocyst (71.8) in Ham's F-10 media were not different from embryos cultured in all other treatments except Ham's F-10 + 10 mug ECGS. A decrease in the percentage of embryos reaching expanded blastocyst (44.7), hatching blastocyst (23.7) and hatched blastocyst (18.4) was observed in Ham's F-10 + 10 mug ECGS. Also, a significant (P<0.01) decrease in development scores at 24, 48 and 72 h was observed for embryos cultured in Ham's F-10 + 10 mug ECGS. No difference was observed in the mean time to reach different developmental stages among treatments. The data suggest that ECGS and FGF at the doses tested have no beneficial effect on early mouse embryo development in vitro and 10 mug of ECGS has inhibitory effect.     

Inhibition of cell proliferation by mechanical agitation involves transient cell cycle arrest at G1 phase in dinoflagellates

Summary.  Cell proliferation of dinoflagellates is negatively affected by mechanical agitation and red tides caused by members of the group have been correlated with periods of calm sea conditions. The mechanism involved in the mechanically transduced inhibition of cell proliferation is thought to involve the disruption of the cell division apparatus. In this study, we used highly synchronized cells and flow cytometry to study the effects of mechanical agitation on cell cycle progression. We observed that mechanical agitation induced transient cell cycle arrest at G₁ phase, in both the heterotrophic dinoflagellate Crypthecodinium cohnii and the photosynthetic dinoflagellate Heteroscapsa triquetra. Received March 12, 2002; accepted July 20, 2002; published online November 29, 2002     

Comparison of growth and cell proliferation kinetics during mouse molar odontogenesis in vivo and in vitro

Abstract. The growth of embryonic first lower mouse molars in vitro was slow and reduced in comparison with in vivo development: the volume of teeth removed on day 15 and 16 of gestation and cultured for 6 days did not exceed the volume reached at day 18 in vivo . The volume of teeth removed on day 17 and 18 and cultured for 6 days either remained constant or decreased. The appearance of post-mitotic odontoblasts and ameloblasts was delayed in vitro . This behaviour might be correlated with a lengthening of the cell cycle ( T _c). In vitro , the average durations of T _c (established by the percentage labelled mitoses technique) were 17.4–20.2 hr and 19.1–19.4 hr for pre-odontoblasts and pre-ameloblasts respectively. In vivo , the corresponding T _c values were 13.9 hr and 13.5 hr. The coordination of mitotic activities of pre-odontoblasts and pre-ameloblasts existing in vivo was maintained in vitro , and therefore seemed to require intra-dental control mechanisms. Non-specific extra-dental serum factors may affect the duration of T _c.     

Regulation of cell growth of a progestin-dependent murine mammary carcinoma in vitro: progesterone receptor involvement in serum or growth factor-induced cell proliferation

Primary cultures of the medroxyprogesterone acetate-induced mouse mammary tumor line C4-HD are stimulated by medroxyprogesterone acetate (MPA) or progesterone. Serum obtained from ovariectomized, MPA-treated animals (OVX-MPA) exerts a stimulatory effect that is significantly higher than that induced by serum obtained from OVX mice with the exogenous addition of MPA, suggesting the involvement of MPA-induced serum factors potentiating the proliferative effect of MPA. The object of this paper is to further explore the stimulatory effect of mouse serum and to investigate the role of aFGF and bFGF on cell proliferation. The role of PR as possible mediators was tested using two different antiprogestins and antisense oligodeoxynucleotides of PR A isoform. Serum was obtained from OVX untreated or MPA-treated mice and was charcoalized and/or heat-inactivated. The effect of MPA or mifepristone at 10 nM concentrations was tested. Charcoalization and heat inactivation exerted a stimulatory effect (P<0.01) when OVX-serum was used. This effect was potentiated by MPA. Charcoalized OVX-MPA serum induced a significant inhibition of cell proliferation that was restored by the exogenous addition of MPA or by heat inactivation. Mifepristone induced an inhibition of ³H-thymidine uptake when OVX-MPA serum was used. These results suggest that serum factors activated by different manipulations may replace the stimulatory effect of MPA. When charcoalized fetal calf serum (chFCS) was used, a higher proliferative activity was obtained using higher serum concentrations. Mifepristone and onapristone 10 nM also inhibited this effect. aFGF and bFGF 100 ng/ml were both able to stimulate ³H-thymidine uptake. MPA exerted an additive effect. Mifepristone 10 nM inhibited bFGF and MPA+bFGF induced cell proliferation. Antisense oligodeoxynucleotides of PR (ASPR) were used to further confirm the participation of PR in the proliferative pathway of these cells. They inhibited serum and bFGF-induced cell proliferation in a specific dose-dependent manner. Our results suggest that PR play a central role in proliferation and suggest the existence of a cross-talk between steroid and growth factor signaling pathways.     

Comparison of growth and cell proliferation kinetics during mouse molar odontogenesis in vivo and in vitro

The growth of embryonic first lower mouse molars in vitro was slow and reduced in comparison with in vivo development: the volume of teeth removed on day 15 and 16 of gestation and cultured for 6 days did not exceed the volume reached at day 18 in vivo. The volume of teeth removed on day 17 and 18 and cultured for 6 days either remained constant or decreased. The appearance of post-mitotic odontoblasts and ameloblasts was delayed in vitro. This behaviour might be correlated with a lengthening of the cell cycle (Tc). In vitro, the average durations of Tc (established by the percentage labelled mitoses technique) were 17.4-20.2 hr and 19.1-19.4 hr for pre-odontoblasts and pre-ameloblasts respectively. In vivo, the corresponding Tc values were 13.9 hr and 13.5 hr. The coordination of mitotic activities of pre-odontoblasts and pre-ameloblasts existing in vivo was maintained in vitro, and therefore seemed to require intra-dental control mechanisms. Non-specific extra-dental serum factors may affect the duration of Tc.     

Control of proliferation in two cell linesin vitro

Summary In two types of cells growing as monolayer cultures (an established line of Chinese hamster cells and a newly obtained strain of Rat Fibroblasts) no evidence is found for proliferation control by contact inhibition of mitosis or by cell-produced chemical inhibitors. Under the particular conditions of these experiments, the intracellular concentration of a multiplication-stimulating factor (MSF) associated with fetal calf serum appears to play a regulating role. This factor is degraded continually by proliferating as well as by non-proliferating, plateau phase cells. A model is proposed in which the intracellular concentration of MSF is governed by: the amount of MSF available to the cell from the culture medium; the permeability and amount of cell membrane accessible for diffusion (or transport) of MSF; and the rate of its degradation. It is hypothesized that mitosis only occurs if the concentration of MSF exceeds a critical value and that this critical value is a characteristic of the cell type. Consistency of the model with existing data concerningin vitro cell proliferation is discussed.This work was supported by Grants CA-04542 and CA-10372 from the National Cancer Institute, National Institutes of Health, U.S. Public Health Service.Biophysics trainee under United States Public Health Service Training Grant GM-00712 from the National Institutes of Health.Holder of a Dernham Senior Fellowship in Oncology from the American Cancer Society, California Division.     

Alteration of neonatal rat parotid gland acinar cell proliferation by guandethidine-induced sympathectomy.

Newborn rats were injected with guanethidine-sulfate (20 micrograms/g body weight) every 48 hr from 12 hr after birth until day 14 (eight injections per animal). The guanethidine treatment resulted in an 86% absolute reduction in cell number in the superior cervical ganglia of 15 day old rats. The cells which remained after guanethidine treatment showed destruction of mitochondria and an extensive decrease in endoplasmic reticulum. Chemical sympathectomy with guanethidine induced a 3.1 hr lengthening of the acinar cell generation cycle time (17.4 hr to 20.5 hr), resulting from a longer G1 period (6.9 hr in the control group as compared to 10.5 hr in the guanethidine-treated group), as well as a cecrease in the mean percentage of [3H]thymidine-labeled acinar cells (22.3 +/- 0.5% to 19.3 +/- 0.5%) and mean acinar cell mitotic index (2.6 +/- 0.2% to 2.1 +/- 0.1%). A circadian rhythm was found to exist in parotid gland acinar cell mitotic activity of 15 day old rats and the amplitude of the rhythm was reduced from 26.5% to 14.9% in guanethidine-treated rats. This study indicates that the diminution of sympathetic influence on the developing parotid gland results in a slight, but significant alteration in acnar cell proliferation.     

mTOR controls ovarian follicle growth by regulating granulosa cell proliferation

We have shown that inhibition of mTOR in granulosa cells and ovarian follicles results in compromised granulosa proliferation and reduced follicle growth. Further analysis here using spontaneously immortalized rat granulosa cells has revealed that mTOR pathway activity is enhanced during M-phase of the cell cycle. mTOR specific phosphorylation of p70S6 kinase and 4E-BP, and expression of Raptor are all enhanced during M-phase. The predominant effect of mTOR inhibition by the specific inhibitor Rapamycin (RAP) was a dose-responsive arrest in the G1 cell cycle stage. The fraction of granulosa cells that continued to divide in the presence of RAP exhibited a dose-dependent increase in aberrant mitotic figures known as anaphase bridges. Strikingly, estradiol consistently decreased the incidence of aberrant mitotic figures. In mice treated with RAP, the mitotic index was reduced compared to controls, and a similar increase in aberrant mitotic events was noted. RAP injected during a superovulation regime resulted in a dose-dependent reduction in the numbers of eggs ovulated. Implications for the real-time regulation of follicle growth and dominance, including the consequences of increased numbers of aneuploid granulosa cells, are discussed.