Induction of anchorage-independent growth by epidermal growth factor and altered sensitivity to type beta transforming growth factor in partially transformed rat kidney cells

A partially transformed cell line (NRK-PT14) was isolated from normal rat kidney (NRK) cells. Like NRK cells, NRK-PT14 cells required epidermal growth factor for anchorage-independent growth, but lost the additional requirement for exogenous type beta transforming growth factor (TGF-beta). Compared to NRK cells, NRK-PT14 cells did not secrete elevated levels of TGF-beta, but exhibited an altered response to this growth factor. Monolayer growth of NRK cells in a serum-free medium was inhibited by TGF-beta, whereas growth of NRK-PT14 cells was stimulated by TGF-beta. In addition, TGF-beta stimulated epidermal growth factor binding to high affinity sites in NRK cells, but decreased epidermal growth factor binding to NRK-PT14 cells during growth of the cells in serum-free medium. These qualitative changes in the response to TGF-beta may be representative of an intermediate stage in the spontaneous transformation of NRK cells.     

Induction and modulation of anchorage-independent growth by platelet-derived growth factor, fibroblast growth factor, and transforming growth factor-beta

Transforming growth factors (TGFs) reversibly induce the anchorage-independent growth of nontransformed cells. TGF activity is often monitored by the growth of normal rat kidney (NRK) fibroblasts in soft agar, and it is known that more than one growth factor is involved in the regulation of their soft agar growth. To more clearly define the growth factors responsible for the soft agar growth of NRK cells, the effects of four growth factors were examined: platelet-derived growth factor (PDGF); TGF-beta; epidermal growth factor (EGF); and fibroblast growth factor (FGF). This study determined that PDGF induces the soft agar growth of NRK cells, in both plasma-supplemented medium and serum-free medium supplemented with FGF, and neither TGF-beta nor an EGF-related growth factor is required for this effect. It was also determined that FGF, which alone does not induce the soft agar growth of these cells, potentiates the responses of NRK cells to various combinations of PDGF, TGF-beta, and EGF. Interestingly, the effect of TGF-beta was found to depend on the growth factor composition of the medium. In the absence of EGF, TGF-beta partially inhibits the soft agar growth response of NRK cells to PDGF, whereas, in the presence of EGF, TGF-beta increases their response to PDGF. These findings indicate that at least four unrelated growth factors regulate the anchorage-independent growth of NRK cells. These findings have important implications for the use of NRK cells to assay TGFs.     

Purification and biological properties of type beta transforming growth factor from mouse transformed cells

Transforming growth factor type beta (TGF beta) has been purified from serum-free culture fluids of transformed mouse L-929 cells which are capable of continual growth in serum-free medium in the absence of any exogenously added polypeptide growth factors. TGF beta has been purified to homogeneity as judged by NH2-terminal amino acid sequence analysis. Analysis of the purified polypeptide by gel electrophoresis indicates that TGF beta is composed of two polypeptide chains of Mr 12,500 cross-linked by disulfide bonds. TGF beta was characterized by its ability to induce anchorage-dependent normal rat kidney (NRK) cells to grow in soft agar in the presence of epidermal growth factor (EGF). TGF beta was also able to enhance both EGF-induced DNA synthesis and cell proliferation on growth-arrested NRK cells in monolayer cultures under serum-free conditions. We also show that in mouse melanoma B-16 cells under serum-free conditions TGF beta stimulates both DNA synthesis in monolayer cultures and anchorage-independent growth in soft agar. Paradoxically, the anchorage-independent growth in the presence of serum of many human cell lines, including melanomas, and mammary, prostatic, vulvar, and lung carcinomas is inhibited by TGF beta at saturating concentrations similar to those that stimulate colony formation of the rodent cell lines L-929 and B-16 under serum-free conditions. The peculiar action of TGF beta is further revealed by the observations that while EGF and TGF beta synergize to induce inhibition of anchorage-independent growth of A-431 human vulvar carcinoma cells, their effects on the anchorage-independent growth of one human lung carcinoma cell line (A-549) and two human prostatic carcinoma cell lines (PC-3 and DU-145) are antagonistic. Moreover, we show that in the rodent and human cell lines TGF beta interacts with specific cellular receptors which may mediate the actions of TGF beta. We conclude that the expression of both TGF beta and TGF beta receptors by L-929 cells and the stimulation of growth of L-929 cells in serum-free medium by TGF beta suggests that TGF beta may be important for maintaining the transformed state of this tumor cell line, and the way in which a cell responds to TGF beta is dependent on the presence or absence of growth factors contained in the serum.     

Spontaneously transformed rat-liver epithelial-cell line producing autocrine and paracrine growth factors.

A spontaneously transformed rat-liver epithelial-cell line that could proliferative in unsupplemented, serum-free medium and be passaged with trypsinization has been established. At the time of writing, the line has undergone more than 100 passages in serum-free culture. This cell line produced an autocrine growth-stimulatory factor (AGSF) and a paracrine growth-stimulatory factor (PGSF). AGSF had a remarkable growth-stimulatory effect on transformed rat-liver epithelial-cells, but had no such effect on non-transformed rat-liver epithelial-cells that could not proliferate in serum-free medium. AGSF did not show a growth-inhibitory or stimulatory effect on normal rat kidney (NRK) cells in monolayer culture, and did not induce anchorage-independent growth in soft-agar culture even with epidermal growth factor (EGF) or transforming growth factor (TGF)-beta. AGSF was protease-sensitive, but heat- and acid-stable. The molecular weight was about 700 Dalton (Da) on size-exclusion chromatography. PGSF showed a growth-stimulatory effect on NRK cells and induced anchorage-independent growth in soft-agar culture without EGF or TGF-beta. On the other hand, PGSF slightly inhibited the growth of the spontaneously transformed rat-liver epithelial cells. PGSF was heat-, protease- and acid-sensitive. The molecular weight was 30 kDa on size-exclusion chromatography.     

Stimulation of anchorage-independent cell growth by endothelin in NRK 49F cells.

Endothelin (ET) is a vasoconstrictor peptide originally isolated from vascular endothelial cells. Recent studies have revealed that ET has many biological functions including growth factor-like activity. The present study aims to clarify whether ET-1 possesses the ability to stimulate anchorage-independent cellular growth, an indicator of factors with transforming activity. We found that NRK 49F cells possess a large number of high-affinity ET-1 receptors; labeled 125I-ET-1 binding was displaced by unlabeled ET-2 in a similar dose response, but in the case of ET-3, 100-fold more was required. Specific 125I-ET-3 binding was undetectable in NRK 49F cells, indicating that ET receptors in NRK 49F cells are ET-1/ET-2 selective. NRK 49F is a cell line which is most commonly used to assay for anchorage-independent cellular growth. Therefore, we explored whether ETs promote anchorage-independent cellular growth in this cell line. ET-1 and ET-2 stimulated NRK colony formation dose dependently in the presence of 1 nM epidermal growth factor (EGF). In contrast, ET-3 did not have colony-stimulating ability. In the presence of EGF, the maximal effect of ET-1 was approximately 90% of that of transforming growth factor-beta. Moreover, in the presence of maximal stimulating concentrations of EGF and transforming growth factor-beta, ET-1 additionally induced colony formation. These results indicate that ET-1 and -2 possess transforming growth factor-like activity for NRK 49F cells. Since ET-1 and -2 increased intracellular calcium levels, this ion may participate in signal transduction pathways by which ET-1 and -2 promote colony formation.     

Transforming growth factor-beta 1 stimulates proliferation of Morris hepatoma cells in serum-free soft agar culture system supplemented with EGF and insulin

The anchorage-independent growth of Morris hepatoma 7777 (MH) cells in serum-free medium was examined. The influence of insulin, epidermal growth factor and transforming growth factor beta 1 (TGF-beta 1) on [3H]thymidine incorporation and colony formation of the investigated cells was described. Contrary to normal rat hepatocytes TGF-beta 1 plus EGF and insulin were found to stimulate MH cells proliferation in presented conditions. A simple, chemically-defined culture system suitable for research on mitogenic peptides was proposed.     

Isolation and characterization of Kirsten murine sarcoma virus-transformed mouse keratinocytes resistant to transforming growth factor beta

BALB/MK (MK) is a continuous murine keratinocyte line whose cells are strictly dependent on exogenous epidermal growth factor (EGF) for growth in culture. A derivative cell, KC, resulted from Kirsten murine sarcoma virus transformation, and these cells no longer require EGF for their growth. Despite differences in MK and KC growth conditions, both cell lines are growth inhibited by picomolar concentrations of transforming growth factor-beta (TGF-beta). When MK and KC cells were maintained in the presence of TGF-beta, resistant variants eventually proliferated only from the KC population. In an attempt to determine the mechanism of development of TGF-beta resistance, the TGF-beta-resistant cells (KCR cells) were compared with TGF-beta-sensitive KC cells with regard to growth properties, TGF-beta 1 binding characteristics, and gene expression. KCR cells continued to synthesize DNA and proliferated in the presence of TGF-beta 1 concentrations up to 2 nM, which was 500-fold greater than the ED50 for the sensitive cells. Although the KCR cells possess similar receptor numbers and affinity for TGF-beta 1, we observed differences in affinity cross-linking studies. The KCR cells expressed more of the type III, high molecular weight cell surface binding protein and less of the type II than the KC cells. The type I moiety was clearly altered to a smaller size in some, but not all, KCR cells. In gene regulation studies, there was no apparent difference in c-Ki-ras and v-Ki-ras mRNA levels in the KC and KCR cells. Additionally, expression of TGF-alpha and TGF-beta 1 mRNA was similar in MK, KC, and KCR cells. The expression of proliferation-associated genes, such as c-myc and MGSA/c-gro/kc, which were markedly decreased by TGF-beta 1 in the MK and KC cells, was not altered by TGF-beta 1 in the KCR cells. The data suggest that the loss of TGF-beta 1 responsiveness in the KCR cells was due to an alteration in the TGF-beta receptor that did not permit signal transduction, although the existence of postreceptor alterations cannot be excluded.     

Modified responsiveness of v-Ha-ras-transfected rat fibroblasts to growth factors and a tumor promoter

The correlation of the phenotypic changes of v-Ha-ras transfected cells with the expression of p21ras and the modified responses to growth factors and a tumor promoter were examined. Transfection of the v-Ha-ras gene together with the neomycin-resistance gene into 208F rat fibroblasts yielded transformed clones characterized by morphological changes, anchorage-independent growth, and tumorigenicity in nude mice. The degrees of these biological alterations were parallel with the expression of mRNA and protein of the ras gene. In ras-transformed cells, anchorage-independent growth was stimulated by epidermal growth factor (EGF), insulin, bombesin, and fibroblast growth factor, whereas in the parental 208F cells, anchorage-independent growth was observed only in the presence of EGF, and there were many fewer EGF-induced colonies than those in the ras-transformed clones. A tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) also augmented anchorage-independent growth of ras-transformed cells and induced morphological changes in monolayer cultures without altering the expression of the ras gene or phosphorylation of the p21ras protein. Retinoic acid inhibited the TPA-induced anchorage-independent growth. These results showed a good correlation of the expression of p21ras with the phenotypic changes and the increased sensitivity of the p21ras-expressing cells to the stimulation of growth factors and tumor promoter.     

Anchorage-independent growth of v-myc-transformed Balb/c 3T3 cells is promoted by platelet-derived growth factor or co-transformation by other oncogenes

Murine fibroblasts transformed by the myc oncogene have a reduced growth factor dependence for both anchorage-dependent and anchorage-independent proliferation. Here we show that v-myc-transformed Balb/c 3T3 cells require, in addition to insulin, only platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) for anchorage-dependent growth in serum-free media. PDGF, however, cannot efficiently be substituted by EGF for anchorage-independent growth. The results suggest that constitutive v-myc expression reduces cellular growth factor requirements by non-autocrine mechanisms for proliferation in monolayer cultures. In contrast, v-myc-transformed cells require plasma components and growth factors of the 'competence' type for anchorage-independent growth. We also demonstrate that the requirement for PDGF by the myc-transformed cells can be abrogated by v-K-ras, v-src and v-fos but not the v-raf oncogene. The results demonstrate that oncogenes can cooperate in the expression of the transformed phenotype by also drastically reducing cellular growth factor requirements.     

Synergistic effect between EGF and TGF-beta1 in inducing oncogenic properties of intestinal epithelial cells

Transforming growth factor (TGF)-beta1 has a biphasic effect on rat intestinal epithelial (RIE) cells. By itself, TGF-beta1 functions as a tumor suppressor by inhibiting the growth, migration and invasion of RIE cells. We show in this study that in conjunction with epidermal growth factor (EGF), TGF-beta1 helped to augment migration, invasion and anchorage-independent growth (AIG) compared to that by EGF alone. EGF plus TGF-beta1 induced a dramatic morphological change characteristic of epithelial-mesenchymal transition (EMT). The mechanism for this enhanced effect of TGF-beta1 and EGF on oncogenic properties was explored by analysis of EGF- and TGF-beta1-mediated signaling pathways and complementary DNA arrays. TGF-beta1 augmented EGF-mediated signaling of mitogen-activated protein kinase (MAPK) and AKT by enhancing and prolonging the activation of the former and prolonging the activation of the latter. Inhibition of MAPK, but not phosphoinositide-3 kinase (PI3K), abolished TGF-beta1 plus EGF-induced EMT and downregulation of E-cadherin at mRNA and protein levels. By contrast, cell migration and invasion were sensitive to inhibition of either MAPK or PI3 kinase. TGF-beta1 plus EGF-induced AIG was significantly more resistant to inhibition of PI3K and MAPK compared to that induced by EGF alone. EGF and TGF-beta1 synergistically induced the expression of a series of proteases including matrix metalloproteinase (MMP) 1 (collagenase), MMP3, MMP9, MMP10, MMP14 and cathepsin. Among them, the expression of MMP1, MMP3, MMP9 and MMP10 was MAPK dependent. Inhibition of the MMPs or cathepsin significantly blocked EGF plus TGF-beta1-induced invasion, but had no effect on colony formation. Phospholipase C (PLC) and Cox2 induced by EGF plus TGF-beta1 also played a significant role in invasion, whereas PLC was also important for colony formation. Our study reveals specific signaling functions and induction of genes differentially required for enhanced effect of EGF- and TGF-beta1-induced oncogenic properties, and helps to explain the tumor-promoting effect of TGF-beta1 in human cancer with elevated expression or activation of TGF-beta1 and receptor protein tyrosine kinases.     

Comparison of growth requirements of two human intratumoral colon carcinoma cell lines in monolayer and soft agarose

The human colon, intratumoral subpopulations HCT 116 and HCT 116a were established in chemically defined medium supplemented with transferrin, insulin, epidermal growth factor (EGF), triiodothyronine, hydrocortisone, and sodium selenite. The responsiveness of the adapted cell lines to these growth factors was compared in anchorage-dependent and -independent assays. HCT 116 cells maintained in serum-free conditions were further adapted to growth factor deprivation, and the effects of these polypeptides were determined in anchorage-independent assays. In monolayer, HCT 116 cells adapted to grow in serum-free medium responded to transferrin but not to EGF or insulin. Similarly adapted HCT 116a cells were, however, insensitive to transferrin addition but manifested a 300 and 500% increase in growth rates with EGF and insulin, respectively. Optimal growth of HCT 116 cells was seen in the presence of insulin and transferrin, while maximum proliferation of HCT 116a cells depended on combined insulin, transferrin, and EGF. In soft agarose, both HCT 116 and HCT 116a subpopulations showed a stringent requirement for transferrin. No combination of growth factors without transferrin supported colony formation. These data suggest that (a) these colon tumor subpopulations may be subject to separate growth controls, and (b) there may be an important role for transferrin in anchorage-independent growth and possibly in the maintenance of malignant characteristics.     

Tumour progression in experimental oral carcinogenesis is associated with changes in EGF and TGF-beta receptor expression and altered responses to these growth factors

This study examined the characteristics of premalignant oral epithelial cell lines derived from non-invasive palatal and lingual mucosa of rats painted with the carcinogen 4-nitroquinoline N-oxide (4NQO) in vivo. In contrast to normal keratinocytes, premalignant epithelial cells had an extended life span, were independent of 3T3 fibroblast support, and expressed variable anchorage independence in gel culture and tumorigenicity in athymic mice. The expression of these functional phenotypes did not correlate with the duration of 4NQO treatment. Keratinocytes from 4NQO-treated tissues predominantly had fewer epidermal growth factor (EGF) receptors than normal controls. The expression of high-affinity EGF receptors paralleled the emergence of the anchorage-independent phenotype and was markedly elevated in tumorigenic cell lines. Cell lines with an extended life span expressed fewer transforming growth factor beta 1 (TGF-beta) receptors than their normal counterparts though the loss of these receptors appeared to be unrelated to either anchorage independence or tumorigenicity. Normal keratinocytes were stimulated and inhibited, in a dose-dependent manner, by EGF and TGF-beta respectively. By contrast, a cell line that was immortal, anchorage dependent and non-tumorigenic showed reduced sensitivity to stimulation by EGF and was inhibited only by high concentrations of TGF-beta. Cells that were immortal, anchorage independent and tumorigenic, however, were refractory to EGF and were inhibited only by high concentrations of TGF-beta. There was no correlation between the expression of EGF or TGF-beta cell surface receptors and the response to ligand binding. The results show that tumour progression in rat oral epithelial cells is associated with a progressive independence of growth factor control. The number and distribution of EGF and TGF-beta receptors may be useful markers in more closely defining the stages of epithelial tumour progression.     

Epidermal growth factor-dependent dissociation of CrkII proto-oncogene product from the epidermal growth factor receptor in human glioma cells.

Human glioma cells frequently overexpress epidermal growth factor receptor (EGFR). We found that the CrkII proto-oncogene product was associated with the EGFR in human glioma cells in the absence of epidermal growth factor (EGF). EGF stimulation of glioma cells induced the phosphorylation of tyrosine 221 of the CrkII protein, which correlates with its dissociation from the EGFR. By contrast, Shc and Grb2 were inducibly associated with the EGFR in response to EGF stimulation of glioma cells. In A431 cells, epidermoid carcinoma cells which overexpress EGFR, CrkII was tyrosine-phosphorylated and associated with the EGFR in an EGF-dependent manner. Therefore, the dissociation of CrkII from the EGFR upon stimulation with EGF appears to be specific to glioma cells. The Cbl oncogene product was also tyrosine-phosphorylated in U87MG glioma cells upon EGF stimulation. However, unlike in other cell lines, CrkII was not inducibly bound to Cbl in U87MG glioma cells. Thus, EGF-dependent binding of CrkII to phosphotyrosine-containing proteins appears to be suppressed in glioma cells. To evaluate the physiological role of dissociation of CrkII from EGFR, we expressed the CrkII-23 mutant in glioma cells. CrkII-23 mutant, which was isolated as a suppressor gene of the EGF-dependent transformation of NRK cells, binds constitutively to EGFR. We found that expression of CrkII-23 inhibited the anchorage-independent growth of the glioma cells in the presence of EGF. Taken together, these data implicate EGF-dependent dissociation of CrkII from EGFR in the oncogenicity of human glioma cells.     

Effect of rous sarcoma virus transformation of rat-1 fibroblasts upon their growth factor and anchorage requirements in serum-free medium

The proliferative response of nontransformed rat embryo (Rat-1) cells and avian sarcoma virus-transformed B31 cells to high-density lipoprotein (HDL), transferrin, insulin, epidermal growth factor (EGF), and fibroblast growth factor has been compared. HDL, added in combination with transferrin, supported the active proliferation of low-density cultures of both Rat-1 and B31 cells. No major difference in the sensitivity of Rat-1 or B31 cells to HDL and transferrin was observed when cells were maintained on dishes coated with an extracellular matrix (ECM) obtained from bovine corneal endothelial cells. The two cell types differed in their response to the other known growth-promoting agents, however, in contrast to Rat-1 cells, transformed B31 cells no longer respond to EGF and fibroblast growth factor and respond only inconsistently to the mitogenic stimulus of insulin. Nontransformed Rat-1 cells and transformed B31 cells grown in the presence of medium containing, respectively, HDL, transferrin, insulin, EGF, and dexamethasone or HDL, transferrin, and insulin could be subcultured for more than 50 generations in the complete absence of serum without significant alteration in morphology, growth rate, or tumorigenicity (B31 cells). When plastic or collagen-coated dishes were used as the substrate instead of ECM-coated dishes, nontransformed Rat-1 cells grew very slowly in the serum-free medium described above. Dishes coated with collagen were not more efficient than was plastic in supporting growth of Rat-1 cells under these conditions. Coating dishes with fibronectin, however, clearly improved their growth, bringing the final cell density of the cultures up to 50% of that obtained on ECM-coated dishes. In contrast, transformed B31 cells grew significantly in serum-free medium when seeded on plastic or collagen-coated dishes, and the final cell density reached by cells on these substrates was 50% of that of cells maintained on ECM-coated dishes. In addition, B31 cells grew equally well when seeded on fibronectin- or ECM-coated dishes. The transformed cells thus showed less stringent substrate requirements when grown under serum-free conditions than did nontransformed Rat-1 cells. Our data also indicate that HDL, in combination with transferrin, supported efficient anchorage-independent growth of B31 cells. Fibroblast growth factor, but not insulin or EGF, further improved anchorage-independent growth of these cells. The capacity of cells to form colonies in semisolid medium when exposed to HDL and transferrin seems to correlate with high tumorigenic potential.     

Transforming growth factor-alpha secretion by epidermal growth factor-dependent human tumor cell lines

Pairs of cell lines from spontaneous human tumors (cervical adenocarcinoma, melanoma, and synovial sarcoma) were established using serum-free culture conditions with and without exogenous epidermal growth factor (EGF). EGF-adapted cultures of melanoma and cervical adenocarcinoma origin secreted higher levels of bioactive transforming growth factor alpha (TGF-alpha) when compared to cultures maintained in the absence of EGF. Depletion of EGF for these EGF-adapted cultures resulted in growth arrest. In contrast, the sarcoma cell lines did not secrete TGF-alpha regardless of the culture conditions but EGF significantly stimulated proliferation of these cells in short-term assays. We show that exogenous EGF induces TGF-alpha production and supports proliferation of tumor cells of various tissue origin but is not essential for in vitro growth factor-deprived conditions.     

cbl-b inhibits epidermal growth factor receptor signaling

The role of cbl-b in signaling by the epidermal growth factor receptor (EGFR) was studied and compared with c-cbl. We demonstrate in vivo, that cbl-b, like c-cbl, is phosphorylated and recruited to the EGFR upon EGF stimulation and both cbl proteins can bind to the Grb2 adaptor protein. To investigate the functional role of cbl proteins in EGFR signaling, we transfected cbl-b or c-cbl into 32D cells overexpressing the EGFR (32D/EGFR). This cell line is absolutely dependent on exogenous IL-3 or EGF for sustained growth. 32D/EGFR cells overexpressing cbl-b showed markedly inhibited growth in EGF compared to c-cbl transfectants and vector controls. This growth inhibition by cbl-b was the result of a dramatic increase in the number of cells undergoing apoptosis. Consistent with this finding, cbl-b overexpression markedly decreased the amplitude and duration of AKT activation upon EGF stimulation compared to either vector controls or c-cbl overexpressing cells. In addition, the duration of EGF mediated MAP kinase and Jun kinase activation in cells overexpressing cbl-b is shortened. These data demonstrate that cbl-b inhibits EGF-induced cell growth and that cbl-b and c-cbl have distinct roles in EGF mediated signaling.     

Effects of transforming growth factor beta on the anchorage-independent growth of murine epithelial JB6 cells

The work described in this paper demonstrates that transforming growth factor beta (TGF-beta) induces the soft agar growth of murine epidermal JB6 clone 41 (Cl 41) cells. In this regard, TGF-beta is more effective than either 12-O-tetradecanoylphorbol-13-acetate or epidermal growth factor. Together, TGF-beta 1 and epidermal growth factor produce a greater stimulation of soft agar growth than either growth factor alone. In contrast, addition of TGF-beta 1 and 12-O-tetradecanoylphorbol-13-acetate together does not stimulate soft agar growth beyond that produced by TGF-beta 1 alone. Interestingly, retinoic acid inhibits the ability of all three factors to induce the anchorage-independent growth of Cl 40 cells. TGF-beta also exerts long-term effects on Cl 41 cells. This was determined by isolating TGF-beta-induced soft agar colonies and examining their dependence on TGF-beta. Five of the six anchorage-independent clones isolated after TGF-beta 1 treatment were found to exhibit anchorage-independent growth in the absence of TGF-beta. In addition, these clones respond far more strongly to TGF-beta 1 than do the parental Cl 41 cells in terms of both the numbers and the sizes of colonies formed in soft agar. The findings reported here are compatible with the proposal that TGF-beta mediates some effects of 12-O-tetradecanoylphorbol-13-acetate.     

Gefitinib ('Iressa', ZD1839), a selective epidermal growth factor receptor tyrosine kinase inhibitor, inhibits pancreatic cancer cell growth, invasion, and colony formation

Pancreatic cancer is a devastating malignancy, characterized by low responsiveness to conventional chemotherapies. Gefitinib, an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) has shown clinical activity against EGFR-expressing tumors. Since pancreatic cancers frequently overexpress EGFR (ErbB-1) and its ligands, our aim was to investigate the potential role of gefitinib in this disease. The GI50 of gefitinib as well as the effects of gefitinib on growth factor actions in pancreatic cancer cell lines were analyzed using MTT assays. FACS analysis using Annexin and propidium iodide (PI) staining were performed to study cell cycle, apoptosis and cell death. Western blot analysis was carried out to investigate expression levels of the 4 members of the ErbB family of receptors in pancreatic cancer cell lines, as well as MAP kinase and EGFR phosphorylation. Soft agar assays were used to measure colony formations. Invasiveness of cancer cells was analyzed using Matrigel-coated filters. gefitinib inhibited cell proliferation of pancreatic cancer cell lines with GI50 concentrations ranging from 2.5 to over 10 micro M. Gefitinib completely inhibited EGF-induced cell proliferation, but did not significantly influence insulin-like growth factor (IGF)-induced mitogenesis. Gefitinib also completely abolished EGF-induced phosphorylation of EGFR and MAP kinase. Furthermore, gefitinib inhibited basal and EGF-induced anchorage-independent cell growth and invasion. Our data demonstrate that gefitinib inhibits pancreatic cancer cell growth through EGFR-dependent pathways. Gefitinib also inhibits anchorage-independent growth and invasiveness, suggesting that gefitinib may offer a new approach for the treatment of pancreatic cancer.     

Differential effects of transforming growth factor beta 1 on the growth of poorly and highly metastatic murine melanoma cells

We have examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the growth of paired murine melanoma cell clones that differ with respect to their experimental metastatic potential. Neither poorly (clone 16) nor highly (clone M2) metastatic cells were capable of anchorage-independent growth in 0.3% agar/Dulbecco's modified Eagle's medium in the absence of serum. However, both clones were capable of anchorage-independent growth in 0.3% agar/Dulbecco's modified Eagle's medium containing 10% calf serum. Colony formation in the presence of 10% calf serum was enhanced in a dose-dependent manner by TGF-beta 1 (half-maximal dose, 0.1 ng/ml) and was 5- to 10-fold greater than colony formation in the presence of 10% calf serum alone. Under anchorage-dependent (monolayer) conditions, neither clone grew in the absence of serum or in medium containing less than 1% calf serum. The monolayer growth of poorly metastatic cells (clone 16) was enhanced in a dose-dependent manner by TGF-beta 1 in medium supplemented with calf serum. Growth was 3.5-fold and 2.3-fold greater than untreated controls after 5 days in submitogenic (0.5%) and mitogenic (10%) concentrations of calf serum, respectively. In contrast, TGF-beta 1 had no effect on the monolayer growth of highly metastatic cells (clone M2) either in submitogenic (0.5%) or mitogenic (10%) concentrations of serum. TGF-beta 1 did not directly stimulate DNA synthesis by either poorly or highly metastatic cells when measured 24 h after TGF-beta 1 treatment. The ability of TGF-beta 1 to stimulate the anchorage-independent growth of metastatic melanoma cells suggests that this potent growth factor may play a role in the growth of these cells in vivo. In addition, the differential sensitivity of poorly and highly metastatic cells to TGF-beta 1 may be relevant to their metastatic potential in vivo. While the mechanism(s) by which TGF-beta 1 stimulates the growth of these cells remains unknown, these differentially metastatic clones of the K-1735 murine melanoma should provide a useful model in which to study the effects of transforming growth factor beta on the metastatic phenotype.     

Growth factor production by human colon carcinoma cell lines

Conditioned media collected under serum-free conditions over 24 to 48 h from 18 human colon adenocarcinoma cell lines were analyzed for transforming growth factor, types alpha and beta (TGF-alpha and -beta), and platelet-derived growth factor in assays for anchorage-independent growth and radioreceptor competition. Detectable levels of TGF-alpha, TGF-beta, and platelet-derived growth factor were produced by 17, 16, and 6 cell lines, respectively. Three liters of conditioned medium from highly tumorigenic (HT-29, DLD-1, and SW620) and nontumorigenic (SKCO-1) colon cell lines and from nonneoplastic rat kidney (NRK-52E) and small intestinal (IEC-6) epithelial cells were purified by high-performance liquid chromatography and assayed for TGF-alpha- and TGF-beta-like activity. The highly tumorigenic colon cell lines produced 10- to 45-fold (soft agar), 19- to 90-fold (radioreceptor), and 4- to 35-fold (radioimmunoassay) more TGF-alpha activity compared to the nonneoplastic rat intestinal (IEC) epithelial cells. NRK-52E did not produce detectable TGF-alpha activity. Radioimmunoassay analysis of peak fractions revealed only TGF-alpha immunoreactivity; epidermal growth factor was not detected. Levels of TGF-beta-like material in the colon carcinoma populations were comparable (HT-29) or elevated (DLD-1, SW620) only 3- to 4-fold (soft agar) or 1- to 3-fold (radioreceptor binding) compared to IEC cells or NRK-52E. Growth factor production is an ubiquitous property of colon carcinoma cell lines maintained in vitro and is consistent with this class of molecule, playing a contributory role in regulating cell growth.