Insulin requirement of human leukemic cell lines

The growth characteristics of human leukemic cell lines in serum supplemented medium and in serum free medium with and without the addition of insulin were investigated. No relation was found between the insulin binding capacity of the cells and their hormone-dependence for growth.     

Clonogenic growth of acute non-lymphocytic leukemia cells in serum-free medium

Summary We devised a serum-free medium for growth of leukemic colony-forming units (CFU-L), enriched with albumin, transferrin, lipids, insulin, hydrocortisone and oligoelements. Blast cells from 15 patients affected by acute non-lymphocytic leukemia were grown in this medium in the presence of human placental conditioned medium obtained under serum-free conditions (sfHPCM). Their clonogenic growth was comparable with that obtained in a serum-containing system. Furthermore, when serum-free cultures were carried out in absence of sfHPCM, either CFU-L growth was prevented or, if clones were obtained, the cultures showed a marked decrease in clonogenicity, indicating their strict dependence on growth factors.     

人结直肠癌细胞系的成球培养方法及转移侵袭能力研究

目的探索人结直肠癌细胞系形成肿瘤细胞球的培养方法及球体内间质化标志物的表达检测。方法结直肠癌细胞系SW480、SW620、LOVO、HT29及HCT116在添加了不同细胞生长因子的无血清培养基（SFM）中悬浮培养,传代更新,诱导分化。免疫荧光技术和RT-PCR分别检测细胞球中间质化标志物N—cadherin及Vimentin的表迭情况。结果5种细胞系均能在特制的SFM中形成细胞球,并稳定的传代增殖。在用血清诱导分化后,又重新贴壁生长,且与亲代细胞保持一致的细胞形态。细胞球中间质化标志物N—cadherin、Vimentin表达均上调。结论结直肠癌细胞系在低黏附及添加了细胞生长因子R—Spondin 1、Noggin的无血清环境中更容易形成悬浮生长的肿瘤细胞球,细胞球细胞比亲代细胞更具有转移侵袭能力。     

Establishment of serum based essentiall free of proteolytic activity for the culture of mouse pancreatic islets.

The present study demonstrates that a) serum based culture medium degrades 125I inhibits its proteolytic activity leading to the recovery of more insulin secreted by islets cultured in the presence of high glucose concentration alone or with glucagon; c) aprotinin also favoured the accumulation of secreted insulin by protecting the hormone from a residual degradative capacity of the hear treated serum.     

Establishment of serum based medium essentially free of proteolytic activity for the culture of mouse pancreatic islets

Summary The present study demonstrates that a) serum based culture medium degrades¹²⁵I insulin; b) heat in-activation of serum (1 h, 56°C) inhibits its proteolytic activity leading to the recovery of more insulin secreted by islets cultured in the presence of high glucose concentration alone or with glucagon; c) aprotinin also favoured the accumulation of secreted insulin by protecting the hormone from a residual degradative capacity of the heat treated serum.This work was supported by a grant (No. 71 5 426-2) from the INSERM and by the CNRS.These results have been presented at the V International Congress of Endocrinology. Hamburg, July 18–24, 1976.Acknowledgments. We should like to thank Mrs E. Gammelgard and K. Christensen for their technical assistance.     

Clonogenic growth of acute non-lymphocytic leukemia cells in serum-free medium

We devised a serum-free medium for growth of leukemic colony-forming units (CFU-L), enriched with albumin, transferrin, lipids, insulin, hydrocortisone and oligoelements. Blast cells from 15 patients affected by acute non-lymphocytic leukemia were grown in this medium in the presence of human placental conditioned medium obtained under serum-free conditions (sfHPCM). Their clonogenic growth was comparable with that obtained in a serum-containing system. Furthermore, when serum-free cultures were carried out in absence of sfHPCM, either CFU-L growth was prevented or, if clones were obtained, the cultures showed a marked decrease in clonogenicity, indicating their strict dependence on growth factors.     

Effect of cell proliferation on the condensation of the X chromosome in culture medium of the individual karyotype 49 XXXXX]

The influence of cell proliferation on the condensation of the X chromosome was observed in vitro in human fibroblasts with 49 XXXXX karyotype. The frequency of cells with four Barr-bodies is low during the logarithmic growth phase and increases to 80% when the cells are becoming confluent or, independently of cell contact, when cell growth is arrested in a medium with low serum content. The condensation of th X chromosomes is reversible when the cells start growing again in medium with a higher serum content.     

Survival of BSC-1 cells through the maintenance of cell volume brought about by epidermal growth factor depends on attachment to the substratum

Summary Addition of epidermal growth factor to culture medium without calf serum suppressed the increase in cell volume and then enhanced the survival of BSC-1 cells attached to culture dishes. However, these effects of epidermal growth factor were not observed in the case of cells on dishes coated with heat-denatured bovine serum albumin.     

Survival of BSC-1 cells through the maintenance of cell volume brought about by epidermal growth factor depends on attachment to the substratum

Addition of epidermal growth factor to culture medium without calf serum suppressed the increase in cell volume and then enhanced the survival of BSC-1 cells attached to culture dishes. However, these effects of epidermal growth factor were not observed in the case of cells on dishes coated with heat-denatured bovine serum albumin.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [3H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [3H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Periimplantation-like growth and development of mouse blastocysts in medium containing horse serum

Horse serum (HS) supported growth and differentiation of blastocysts as well as or better than fetal bovine serum (FBS) (p less than 0.01) as measured by a) the onset of trophoblastic outgrowth, b) the size of the resultant outgrowths, c) the size of nuclei in trophoblasts after their outgrowth and d) the size of the inner cell mass in outgrowths. Moreover, polyamines were found to be approximately 10-100 times less toxic to embryos when added to medium containing HS than when the medium contained FBS.     

Effect of amniotic fluid and fetal bovine serum on the morphogenesis of mouse duodenal villi in organ culture

Summary When duodenal explants of 15-day mouse foetuses are cultured in Trowell T8 medium during 72 h, villi do not develop. If mouse amniotic fluid (25%) is added to the same medium, short villi appear after 12 h of culture and medium size villi are seen at 48 h. Bovine amniotic fluid and fetal bovine serum promote cell growth but morphogenesis of intestinal villi is far less stimulated.Supported by grand MA-6069 from the Medical Research Council of Canada.     

Effect of cell proliferation on the condensation of X chromosomes in the form of Barr-bodies, in human 46 XX fibroblast cultures with different serum concentrations]

The frequency of Barr-bodies in cultivated 46 XX human fibroblasts considerably increases when in confluent monolayer cultures the cells no longer divide. Or, independently of cell contact, when the cells are grown in medium with low serum content, which will slow down or entirely arrest further growth. The frequency of Barr-bodies again diminishes when after the administration of culture medium with sufficient serum concentration, the cells start growing again, indicating that the condensation of the X chromosome is reversible under control of the cell proliferation.     

Effect of low density lipoprotein on proteoglycan synthesis by aorta cells in culture.

Increasing the content of human serum low density lipoprotein in the growth medium led to greater incorporation of 35S-sulfate into proteoglycan (mostly into dermatan sulfate) by primary aorta cells but did not affect similar incorporation by fibroblast cells. These results suggest a mechanism which can explain the increased deposition of lipid in aorta due to hyperlipidemia.     

Hormone content of mouse pancreatic islets subjected to different in vivo and in vitro functional demands

Summary Mice treated for 4 days with tolbutamide displayed decreased serum glucose values with a concomitant decrease of their islet insulin content. Mouse islets cultured for 1 week at a low (3 mM) or a high (28 mM) glucose concentration contained less insulin than non-cultured islets and islets cultured at a medium (11 mM) glucose concentration. All groups of cultured islets contained more glucagon than non-cultured islets. The somatostatin content of high- and medium-glucose cultured islets was higher than that of freshly isolated islets.The skilled technical assistance of Astrid Nordin, Ewa Forsbeck and Eva Törnelius is gratefully acknowledged. Financial support was received from the Swedish Medical Research Council (12X-109; 12X-2297), the Nordic Insulin Fund and the Swedish Diabetes Association.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Summary Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptornegative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [³H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10^–6 M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10^–5 M linoleic acid or 10^–5 M arachidonic acid but not by 10^–6 M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (K_d 0.54 nM±0.11 nM; n=6) than growth in medium supplemented with untreated serum (complete medium) (K_d=1.68 nM±0.48 nM; n=6) (p<0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10^–5 M linoleic acid or 10^–5 M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10^–5 M stearic acid or 10^–5 M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10^–5 M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [³H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [³H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

无血清分离人结直肠癌细胞的体外培养形态及标志物表达特征

目的探讨人结直肠肿瘤干细胞在体外分化过程中细胞形态和干细胞相关标志物CD133的表达变化,为进一步研究结直肠肿瘤干细胞分化走向提供实验依据。方法取来源于人结直肠癌的细胞系HCT116,无血清培养分离出CD133+细胞,加血清诱导分化,相差显微镜下观察其形态变化;在未分化状态下无血清培养第7天和14天与血清诱导分化后收集细胞,利用流式细胞仪检测干细胞标志物CD133的表达量,采用激光共聚焦检测CD133表面标记分子的表达。结果 1)细胞形态:无血清培养分离的CD133+细胞,在生长过程中聚集成规则的细胞球,血清诱导后即贴壁生长,贴壁形态与同来源细胞形态一致,且再次无血清悬浮培养后聚集成球稳定生长。2)标志物变化:结直肠肿瘤干细胞未分化时CD133表面标记分子高表达,流式细胞仪检测未分化细胞CD133第7天表达率为(20.4±0.52)%,第14天表达率为(78.5±2.80)%,分化后表达率为(0.50±0.17)%。结论细胞形态和标志物表达改变均表明高表达CD133+的HCT116结直肠癌肿瘤干细胞可定向分化为同源的结直肠癌细胞,CD133+细胞经血清诱导后表达下调而使细胞失去干细胞特性。     

Retention ofB. burgdorferi pathogenicity and infectivity after multiple passages in a co-culture system

In vitro cultivation ofB. burgdorferi in BSK medium results in the loss of infectivity and pathogenicity after repeated passages. To prevent this loss, a feeder layer of tibio-tarsal joint tissue derived from newborn LEW/N rats was grown on Cytodex 3 microcarriers in ESG (formerly BSKE), a novel medium developed to support the growth of both the feeder layer andB. burgdorferi. A new pathogenic isolate (FNJ) and a high passage, non-pathogenic strain (TNJ) grew well in this co-culture system with high yields of viable organism. FNJ caused no growth inhibition or visible damage to the cells in the feeder layer. FNJ remained arthritogenic for newborn LEW/N rats after 22 passages in the co-culture system, but lost its arthritogenicity after 7 passages when cultured in BSK medium. This borrelia-mammalian tissue co-culture technique presents an experimental system to study the long term interactions ofB. burgdorferi with the infected host tissues in vitro, as well as facilitate diagnostic tests and vaccine development.This article forms New Jersey Agricultural Experiment Station paper no. D8420-19-92.     

Fehlende immunogene Wirkung exogenen Insulins bei Hunden nach langsamer Adaptation an Insulin

Summary Subcutaneous injection of exogenous insulin always results in a production of antibodies against insulin. But after a slowly increased dose of insulin (a daily end dose of 3.5–4.2 U/kg) insulin antibodies could not be found in the serum of 2 dogs. This failure of immunogenic action of insulin is discussed as an induction of immunotolerance by the modus of initial antigen application.     

Insulin acts on the hypothalamic glucose-facilitated neurons to induce hyperglycemia and hyperinsulinemia in the rat

Microinjection of insulin (0.04-0.12 IU/microliter) into the anterior hypothalamus or the lateral hypothalamus, but not the ventromedial hypothalamus of the rat brain, caused a dose-dependent rise in blood glucose and in serum insulin. The majority (71.5%) of the glucose-facilitated neurons recorded in the lateral hypothalamic area were excited by intracerebral injection of insulin. The data indicate that insulin acts on the hypothalamic glucose-facilitated neurons to induce hyperglycemia and hyperinsulinemia. It is unknown whether insulin normally reaches the hypothalamic area, or how it might do so.