Serum Cell Lines Research Articles

Stem Cell Media Culture of Melanoma Results in the Induction of a Nonrepresentative Neural Expression Profile

The ability of cell lines to accurately represent cancer is a major concern in preclinical research. Culture of glioma cells as neurospheres in stem cell media (SCM) has been shown to better represent the genotype and phenotype of primary glioblastoma in comparison to serum cell lines. Despite the use of neurosphere-like models of many malignancies, there has been no robust analysis of whether other cancers benefit from a more representative phenotype and genotype when cultured in SCM. We analyzed the growth properties, transcriptional profile, and genotype of melanoma cells grown de novo in SCM, as while melanocytes share a common precursor with neural cells, melanoma frequently demonstrates divergent behavior in cancer stem cell assays. SCM culture of melanoma cells induced a neural lineage gene expression profile that was not representative of matched patient tissue samples and which could be induced in serum cell lines by switching them into SCM. There was no enrichment for expression of putative melanoma stem cell markers, but the SCM expression profile did overlap significantly with that of SCM cultures of glioma, suggesting that the observed phenotype is media-specific rather than melanoma-specific. Xenografts derived from either culture condition provided the best representation of melanoma in situ. Finally, SCM culture of melanoma did not prevent ongoing acquisition of DNA copy number abnormalities. In conclusion, SCM culture of melanoma does not provide a better representation of the phenotype or genotype of metastatic melanoma, and the resulting neural bias could potentially confound therapeutic target identification.

118 Responses of human pancreatic tumour cells to treatment with anti-EGFR mAb ICR62 and the irreversible EGFR/HER1 and HER2 tyrosine kinase inhibitor BIBW2992

Activation of the insulin-like growth factor-1 receptor (IGF-1R) by IGF-1 and IGF-2 plays a prominent role in the growth and survival of small cell lung cancer (SCLC) by potently activating the PI3K-Akt signal transduction pathway, which is also an important factor in the resistance of SCLC to chemotherapy. A12 is a fully human monoclonal antibody directed against the human IGF-1R that does not cross-react with the insulin receptor. In this study we have utilized A12 to determine the effects of selective antibody-mediated blockade of the IGF-1R on SCLC cell lines. Incubation with A12 resulted in a dose-dependent inhibition of IGF-1-stimulated IGF-1R and Akt activity, with maximal inhibition of approximately 75% at a concentration of 10 μg/ml in the H526 cell line. Growth of the H526 and H146 cell lines in serum was inhibited by a maximum of 50–70% in a dose-dependent fashion, which correlated well with the extent of Akt inhibition. However, growth of the H69 and WBA cell lines was unaffected by A12. Despite almost complete inhibition of IGF-1R phosphorylation by A12, Akt activity remained constitutively high in these cell lines. H526 transfectants expressing a constitutively active Akt allele also were resistant to A12. Treatment with A12 additively enhanced response to carboplatin in the H526 and H146 cell lines but had no effect on the H69 and WBA cell lines. Treatment of the H526 cell line with a combination of A12 and rapamycin was highly synergistic. These data suggest that growth inhibition and chemosensitization of SCLC by A12 is directly correlated with the ability to inhibit PI3K-Akt signaling, with those cell lines showing constitutive PI3K-Akt signaling displaying a high level of resistance to IGF-1R targeted therapy.

Parameters influencing derivation of embryonic stem cells from murine embryos

The derivation of ES cells is poorly understood and varies in efficiency between different strains of mice. We have investigated potential differences between embryos of permissive and recalcitrant strains during diapause and ES cell derivation. We found that in diapause embryos of the recalcitrant C57BL/6 and CBA strains, the epiblast failed to expand during the primary explant phase of ES cell derivation, whereas in the permissive 129 strain, it expanded dramatically. Epiblasts from the recalcitrant strains could be expanded by reducing Erk activation. Isolation of 129 epiblasts facilitated very efficient derivation of ES cell lines in serum- and feeder-free conditions, but reduction of Erk activity was required for derivation of ES cells from isolated C57BL/6 or CBA epiblasts. The results suggest that the discrepancy in ES cell derivation efficiency is not attributable merely to variable prodifferentiative effects of the extra-embryonic lineages but also to an intrinsic variability within the epiblast to maintain pluripotency.

Inhibitors of Sterol Biosynthesis as Staphylococcus aureus Antibiotics

Inhibitors of Sterol Biosynthesis as <i>Staphylococcus aureus</i> Antibiotics

Selective inhibition of SCLC growth by the A12 anti-IGF-1R monoclonal antibody correlates with inhibition of Akt

Activation of the insulin-like growth factor-1 receptor (IGF-1R) by IGF-1 and IGF-2 plays a prominent role in the growth and survival of small cell lung cancer (SCLC) by potently activating the PI3K-Akt signal transduction pathway, which is also an important factor in the resistance of SCLC to chemotherapy. A12 is a fully human monoclonal antibody directed against the human IGF-1R that does not cross-react with the insulin receptor. In this study we have utilized A12 to determine the effects of selective antibody-mediated blockade of the IGF-1R on SCLC cell lines. Incubation with A12 resulted in a dose-dependent inhibition of IGF-1-stimulated IGF-1R and Akt activity, with maximal inhibition of approximately 75% at a concentration of 10mug/ml in the H526 cell line. Growth of the H526 and H146 cell lines in serum was inhibited by a maximum of 50-70% in a dose-dependent fashion, which correlated well with the extent of Akt inhibition. However, growth of the H69 and WBA cell lines was unaffected by A12. Despite almost complete inhibition of IGF-1R phosphorylation by A12, Akt activity remained constitutively high in these cell lines. H526 transfectants expressing a constitutively active Akt allele also were resistant to A12. Treatment with A12 additively enhanced response to carboplatin in the H526 and H146 cell lines but had no effect on the H69 and WBA cell lines. Treatment of the H526 cell line with a combination of A12 and rapamycin was highly synergistic. These data suggest that growth inhibition and chemosensitization of SCLC by A12 is directly correlated with the ability to inhibit PI3K-Akt signaling, with those cell lines showing constitutive PI3K-Akt signaling displaying a high level of resistance to IGF-1R targeted therapy.

The insulin-like growth factor-I (IGF-I) receptor kinase inhibitor NVP-ADW742, in combination with STI571, delineates a spectrum of dependence of small cell lung cancer on IGF-I and stem cell factor signaling

Stem cell factor (SCF)/Kit and insulin-like growth factor-I (IGF-I)/IGF-I receptor (IGF-IR) autocrine loops play a prominent role in the growth of small cell lung cancer (SCLC). Previous data suggested that IGF-I protects cells from apoptosis induced by STI571, an efficient inhibitor of Kit signal transduction, by activating the critical phosphatidylinositol 3-kinase-Akt pathway. To determine if inhibition of IGF-IR signaling would be therapeutically relevant in SCLC, the activity of a novel kinase inhibitor of IGF-IR, NVP-ADW742 (Novartis Pharma AG, Basel, Switzerland), was characterized. Pretreatment of the H526 cell line with NVP-ADW742 inhibited IGF-IR signaling and growth with IC(50) values between 0.1 and 0.4 micro M. SCF-mediated Kit phosphorylation and Akt activation were inhibited with IC(50) values in the 1-5 micro M range. However, NVP-ADW742 affected neither hepatocyte growth factor-mediated Akt activation nor activity of constitutively active Akt. The therapeutic potential of NVP-ADW742 was assessed by determining its effect on growth of several SCLC cell lines in serum. These studies clearly delineated two populations of cell lines as determined by differential sensitivity to NVP-ADW742. One population, which lacks active SCF/Kit autocrine loops, was inhibited with IC(50) values between 0.1 and 0.5 micro M. A second population, which has active SCF/Kit autocrine loops, was inhibited with IC(50) values in the 4-7 micro M range. When these cell lines were treated with a combination of STI571 and NVP-ADW742, no advantage was seen in the former group, whereas, in the latter group, a clearly synergistic response to the combination was seen when growth, apoptosis, or Akt activation was assessed. These data demonstrate that NVP-ADW742 is a potent and selective IGF-IR kinase inhibitor that can efficiently inhibit the growth of cells that are highly dependent on IGF-I signaling. However, for optimal growth inhibition of SCLC cells with an active SCF/Kit autocrine loop, a combination of a Kit inhibitor (STI571) and an IGF-IR inhibitor (NVP-ADW742) appears to be necessary. These observations suggest that, in tumors in which critical signal transduction pathways can be activated by alternative receptors, optimal therapy may require inhibition of multiple receptors.

Effect of M20 interleukin-1 inhibitor on normal and leukemic human myeloid progenitors

This study aimed to assess the effect of the M20 interleukin-1 (IL-1) inhibitor on normal and leukemic hematopoietic cells. The M20-derived IL-1 inhibitor was found to inhibit the growth of various hematopoietic cells. The in vitro proliferation of myeloid cell lines in serum- containing medium or proliferation of these cells induced by IL-1 in serum-free medium (measured by 3H-TdR) were inhibited by the M20 IL-1 inhibitor. In addition, growth of normal progenitors and fresh leukemic cells stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) (as measured by colony and liquid systems) was also inhibited by this factor. After the removal of the IL-1 inhibitor at the peak of growth inhibition, leukemic and normal progenitor cells retain their ability to grow and develop into GM-CSF colonies. These results show that the growth inhibition phenomena were reversible and did not result from a cytotoxic effect. Our data suggest that the M20-derived IL-1 inhibitor might function as a true negative growth regulator of normal and leukemic hematopoietic cells.

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.

Time course study of L-tyrosine aminotransferase induction in rat liver cell lines.

The enhancement of L-tyrosine aminotransferase activity by dexamethasone, an exclusive function of the liver, was serially measured at different passages of eight rat liver epithelial cell lines initiated and continuously grown in either a serum-supplemented medium or a serum-free medium. The enzyme basal activity was found to be 5.4 +/- 1.8 mU for cell lines in serum and 6.8 +/- 3.4 mU for cell lines without serum. Under the influence of dexamethasone (10(-6) mol/l for 5 hours) this basal level could be increased up to 2.9 fold in the presence of serum and 2.5 fold in its absence when investigations were carried out at early passages. During the following subcultures the induction ratio gradually declined and scarcely any induction could be detected after the 15th passage for cells grown in serum and after the 25th passage for cell lines grown without serum.

Effect of Serum on the Yield of Lymphocytic Choriomeningitis Virus in Baby Hamster Kidney Cells

The yields of the Armstrong and WE strains of lymphocytic choriomeningitis virus in baby hamster kidney (BHK) cells cultivated in either bovine, calf, fetal bovine, or horse serum were investigated. Lines of BHK cells were established in these sera. When the infected cell lines were observed by immunofluorescence, the per cent fluorescing cells for a given virus strain did not vary. However, for both strains, the extracellular virus yields per cell were significantly greater in the fetal bovine-cell line than in the other serum-cell lines.