The role of ras gene expression and transforming growth factor alpha production in the etiology and progression of rodent and human breast cancer.
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Animals↗
Biomedical subjects
F Ciardiello
Publications and source records attributed to F Ciardiello.
Expression of transforming growth factor alpha (TGF alpha) in breast cancer.
Transforming growth factor alpha (TGF alpha) is one growth factor that has been circumstantially implicated in regulating the autocrine growth of breast cancer cells. Expression of TGF alpha can be modulated by activated cellular protooncogenes such as ras and by estrogens. For example, the epidermal growth factor (EGF)-responsive normal NOG-8 mouse and human MCF-10A mammary epithelial cell lines can be transformed with either a point-mutated c-Ha-ras protooncogene or with a normal or point-mutated c-neu (erbB-2) protooncogene. In ras transformed NOG-8 and MCF-10A cells but not in neu transformed cells there is a loss in or an attenuated response to the mitogenic effects of EGF. This response may be due in part to an enhanced production of endogenous TGF alpha that is coordinately and temporally linked to the expression of the activated ras gene and to the acquisition of transformation-associated properties in these cells. TGF alpha mRNA and TGF alpha protein can also be detected in approximately 50-70% of primary human breast tumors. In addition, approximately 2- to 3-fold higher levels of biologically active and immunoreactive TGF alpha can also be detected in the pleural effusions from breast cancer patients as compared with the TGF alpha levels in the serous effusions of noncancer patients. Over-expression of a full-length TGF alpha cDNA in NOG-8 and MCF-10A cells is capable of transforming these cells. Finally, expression of TGF alpha mRNA and production of biologically active TGF alpha protein is also found in normal rodent and human mammary epithelial cells.
Animals↗
Expression of cripto, a novel gene of the epidermal growth factor family, in human gastrointestinal carcinomas.
The expression of mRNA for cripto gene, a novel transforming gene of the epidermal growth factor family, was examined in 20 alimentary tract carcinoma cell lines, 60 surgically resected tumor tissues and their adjacent normal mucosas. Although the cripto mRNA was not detected in esophageal carcinomas or in normal mucosas, it was detected in gastric and colorectal carcinomas. In gastric carcinomas, 2.2 kb cripto mRNA was detected in one cell line, all the gastric carcinoma tissues and their adjacent normal mucosas. Of 23 gastric tumor tissues 8 (34.8%) exhibited a higher mRNA level than normal gastric mucosas. cripto mRNA was detected in 2 out of 6 colorectal carcinoma cell lines. Interestingly, 18 (81.8%) out of 22 colorectal carcinoma specimens expressed a higher level of cripto mRNA than that in normal mucosas. The level of the expression was higher than that in gastric carcinoma tissues. The expression was also correlated to tumor stage of colorectal carcinomas.
Adult↗
Regulation by estrogen through the 5'-flanking region of the transforming growth factor alpha gene.
Expression of transforming growth factor alpha (TGF alpha) mRNA and protein can be stimulated by estrogens such as 17 beta-estradiol (E2) in estrogen-responsive rodent and human breast cancer cells. To ascertain if E2 can directly regulate TGF alpha expression through the 5'-flanking region of the human TGF alpha gene, E2-responsive MCF-7 or ZR-75-1 human breast cancer cells or E2-nonresponsive MDA-MB-231 breast cancer cells were transiently transfected with a plasmid containing an 1140-base pair (bp) Sac-I fragment of the TGF alpha 5'-flanking region ligated to the chloramphenicol acetyltransferase (CAT) gene. Cells that were transfected and subsequently treated with physiological concentrations of E2 (10(-11)-10(-8) M) for 24 h exhibited a 2- to 10-fold increase in CAT activity. The E2 stimulation of CAT activity was dose-dependent with an increase first found at 10(-10) M E2. The increase in CAT activity could be detected within 24-36 h after the addition of E2. There was no significant change in CAT activity in transiently transfected MDA-MB-231 cells as mediated through the TGF alpha 5'-flanking region after E2 treatment. MCF-7 cells were also transiently transfected with different fragments of the TGF alpha 5'-flanking region ligated to the luciferase gene. In the absence of E2 treatment, no detectable luciferase activity was found.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Constitutive expression of transforming growth factor alpha does not transform rat thyroid epithelial cells.
To evaluate the role of transforming growth factor alpha (TGF alpha) in transformed rat thyroid epithelial cells, expression and production of TGF alpha were measured in a normal rat thyroid epithelial cell line, FRTL-5, and in the same cells transformed by the Ki-ras oncogene. FRTL-5 cells transformed with Ki-ras exhibit a 5- to 7-fold increase in the levels of TGF alpha-specific mRNA and a 3- to 4-fold enhancement of the amounts of TGF alpha protein in the conditioned medium, as compared with the normal thyroid cells. Although conditioned medium from the Ki-ras transformed FRTL-5 cells or authentic epidermal growth factor or TGF alpha are able to stimulate the anchorage-dependent growth of nontransformed FRTL-5 cells, neither conditioned medium nor the growth factors are able to induce the anchorage-independent growth of these cells in soft agar. FRTL-5 cells were then transfected with an expression vector plasmid containing the human TGF alpha cDNA to directly ascertain if over-expression of this growth factor is able to induce transformation in these cells. The TGF alpha-transfected FRTL-5 clones constitutively produced high amounts of TGF alpha at a level equivalent to or greater than the level found in the conditioned medium from the Ki-ras transformed FRTL-5 clones. Moreover, in contrast to the Ki-ras transformed cells, the TGF alpha transfectants were unable to grow in soft agar, did not form tumors in nude mice, and showed no reduction in the secretion of thyroglobulin. These data demonstrate that unlike Ki-ras, the constitutive expression of biologically active TGF alpha is not entirely sufficient to elicit a transformed phenotype in these cells.
Animals↗
Over-expression of the epidermal growth factor receptor in human breast cancer cells fails to induce an estrogen-independent phenotype.
An association exists in primary human breast tumors between high epidermal growth factor receptor (EGFR) expression and a reduced number or even absence of estrogen receptors (ER). To determine whether an increase in EGFR expression might alter the estrogen responsiveness of an ER-positive human breast cancer cell line, ZR 75-1 cells were cotransfected with a plasmid containing the full-length cDNA for the human EGFR under the transcriptional control of the Harvey murine sarcoma virus (HaMSV) long terminal repeat (LTR) and with a pSV2neo plasmid. Two of the isolated G418-resistant clones were found to constitutively express EGFR levels 15- to 60-fold higher than those found on nontransfected ZR 75-1 cells. The EGFR in these clones were functionally normal since EGF could increase their autophosphorylation and since EGF could enhance the transphosphorylation of p185erbB-2. No change was seen in either the number or affinity of ER in these clones. In addition, the ability of estrogen to stimulate the anchorage-dependent and anchorage-independent growth of these clones was not significantly modified. These results suggest that an increase in EGFR expression alone is not sufficient to induce a hormone-independent phenotype in vitro in human breast cancer cells.
Blotting, Northern↗
8-Chloro-cAMP inhibits transforming growth factor alpha transformation of mammary epithelial cells by restoration of the normal mRNA patterns for cAMP-dependent protein kinase regulatory subunit isoforms which show disruption upon transformation.
Differential regulation of the regulatory subunits of cAMP-dependent protein kinase isozymes correlates with the growth inhibitory effect of site-selective 8-Cl-cAMP demonstrated in cancer cell lines (Ally, S., Tortora, G., Clair, T., Grieco, D., Merlo, G., Katsaros, D., Ogreid, D., Døskeland, S.O., Jahnsen, T., and Cho-Chung, Y.S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6319-6322). Such selective modulation of protein kinase isozyme regulatory subunits was also found in the 8-Cl-cAMP-induced inhibition of both transformation and transforming growth factor alpha (TGF alpha) production in Ki-ras-transformed rat kidney fibroblasts (Tortora, G., Ciardiello, F., Ally, S., Clair, T., Salomon, D. S., and Cho-Chung, Y. S. (1989) FEBS Lett. 242, 363-367). In this work, we have demonstrated that 8-Cl-cAMP antagonizes the TGF alpha effect in TGF alpha-transformed mouse mammary epithelial cells (NOG-8TFC17) at the level of gene expression for cAMP receptor protein isoforms, RI and RII (the regulatory subunits of protein kinase isozymes). Northern blot analysis demonstrated that in the transformed NOG-8TFC17 cells, compared with the nontransformed counterpart NOG-8 cells, the mRNA levels for the RI alpha cAMP receptor protein markedly increased, whereas the mRNA levels for the RII alpha and RII beta cAMP receptor proteins decreased. 8-Cl-cAMP, which induced growth inhibition and phenotypic reversion in NOG-8TFC17 cells, caused an inverse change in the mRNA patterns of the cAMP receptor proteins; RI alpha cAMP receptor mRNA sharply decreased to levels comparable with that of the nontransformed NOG-8 cells, whereas RII beta mRNA increased to a level even greater than that in the NOG-8 cells. In addition, one mRNA species of RII alpha increased, whereas the other RII alpha mRNA species decreased during the treatment. The mRNA level for the catalytic subunit of protein kinase, however, did not change during 8-Cl-cAMP treatment. In addition, 8-Cl-cAMP brought about a reduction in both TGF alpha mRNA and protein levels. These coordinated changes in the expression of the cAMP receptor proteins and TGF alpha were not observed during cis-hydroxyprolineor TGF beta-induced growth inhibition of the NOG-8TFC17 cells. Thus, the antagonistic effect of 8-Cl-cAMP toward TGF alpha-induced transformation involves modulation of the expression of a specific set of cellular genes.
8-Bromo Cyclic Adenosine Monophosphate↗
Differential growth factor expression in transformed mouse NIH-3T3 cells.
The expression of growth factor-specific mRNA transcripts and the presence of biologically active growth factors in the conditioned medium and in the cell extracts from mouse NIH-3T3 cells transformed by different oncogenes (Ki-ras, mos, src, fms, fes, met, and trk), by a DNA tumor virus (SV40), or by a chemical carcinogen (N-nitrosomethylurea) were studied. In contrast to NIH-3T3 cells or simian virus 40 (SV40)-transformed 3T3 cells, all the other transformed NIH-3T3 cell lines expressed a 4.5 kb transforming growth factor-alpha (TGF alpha)-specific mRNA transcript and secreted immunoreactive and biologically active TGF alpha ranging from 100 to 225 ng/10(8) cells/48 h. In addition, in the transformed cell lines that were secreting elevated levels of biologically active TGF alpha, there was a 75-95% reduction in the total number of epidermal growth factor receptors on these cells. A 2.6 kb TGF beta mRNA transcript and TGF beta protein in the conditioned medium (30-140 ng/10(8) cells/48 h) was also detected in those lines expressing TGF alpha. Basic fibroblast growth factor-like activity (11-50 ng/10(8) cells) was detected in the cell lysates from NIH-3T3 cells transformed with N-nitrosomethylurea or with trk, where expression of specific 6.9 and 3.9 kb mRNA transcripts for basic fibroblast growth factor could also be found. B chain (c-sis) expression of platelet-derived growth factor was present only in trk-transformed NIH-3T3 cells in which specific c-sis 6.5 and 4.6 kb transcripts were identified. In contrast, platelet-derived growth factor A chain expression of 2.9 and 2.3 kb transcripts was found in ras-, met-, mos-, and fms-transformed NIH-3T3 cells. These results suggest that the expression of different sets of growth factors is controlled in part by structurally distinct groups of transforming genes.
Animals↗
Stromal influences on transformation of human mammary epithelial cells overexpressing c-myc and SV40T.
The proto-oncogene c-myc and the oncogene SV40T, both of which have been implicated in the process of cellular immortalization in vitro, have been introduced via amphotropic retroviral expression vectors into the human mammary epithelial cell (HMEC) line 184A1N4 (A1N4). Two stable cell lines were established by growth in selective medium and were found to overexpress either c-myc (A1N4-myc) or SV40T antigen (A1N4-T). Neither the A1N4, A1N4-myc, or A1N4-T cells will grow in soft agar or form tumors in nude mice. However, A1N4-T or A1N4-myc cells, but not the parental A1N4 cells, form colonies in soft agar in response to either epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), or basic fibroblast growth factor (bFGF). Like EGF and TGF alpha, bFGF is moderately mitogenic for the anchorage-dependent growth (ADG) of all three cell lines. Further, co-cultivation of A1N4-T or A1N4-myc cells with primary diploid mammary fibroblasts can also induce the anchorage-independent growth (AIG) and stimulate the ADG of A1N4-T or A1N4-myc. In addition, conditioned medium obtained from these mammary fibroblasts also stimulated the AIG of the A1N4-T and A1N4-myc cells and was found to contain immunoreactive TGF alpha and bioactive FGF. The mammary fibroblasts express specific mRNA transcripts for bFGF and acidic FGF (aFGF). These results suggest that growth factors such as TFG alpha or FGF, which may be derived from the adjacent mammary stroma, might influence in a paracrine manner the phenotypic characteristics of a population of human mammary epithelial cells toward transformation.
Agar↗
Transforming growth factor-alpha messenger RNA localization in the developing adult rat and human mammary gland by in situ hybridization.
Transforming growth factor-alpha (TGF alpha) has been implicated in the autocrine growth control of a number of different rodent and human tumor cells, including breast cancer cells. Although TGF alpha has been detected in a limited number of normal tissues, its distribution and physiological function in the mammary gland are relatively unknown. TGF alpha mRNA expression was detected by in situ hybridization with a labeled TGF alpha antisense RNA probe and quantitated by application of computer-assisted digital image processing in both the ductal and alveolar epithelial cells in the virgin rat and nulliparous and parous human mammary glands. During pregnancy and lactation, the level of TGF alpha mRNA expression in the ductal and alveolar epithelial cells increased two- to threefold, while a heterogeneous yet strong expression of TGF alpha mRNA could also be detected in approximately 10-15% of the surrounding stromal cells in the pregnant mammary gland.
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Epidermal growth factor receptor, but not c-erbB-2, activation prevents lactogenic hormone induction of the beta-casein gene in mouse mammary epithelial cells.
The HC11 cell line was isolated from mammary gland cells of pregnant mice. The cells displayed a normal phenotype and retained some characteristics of mammary epithelial cell differentiation. After treatment with the lactogenic hormones prolactin and glucocorticoids, the HC11 cells expressed the milk protein beta-casein. Various oncogenes were transfected and expressed in HC11 cells. The oncogenes were tested for their transformation ability and for their effects upon the differentiation of the HC11 cells. All of the oncogenes tested, including activated human Ha-ras, human transforming growth factor-alpha, activated rat neuT, and human c-erbB-2 activated by a point mutation in the transmembrane domain, caused transformation of the HC11 cells, as shown by tumor formation in nude mice. HC11 cells expressing the neuT and activated c-erbB-2 genes synthesized beta-casein in response to lactogenic hormones, whereas those expressing the Ha-ras or transforming growth factor-alpha oncogenes were no longer able to respond to the lactogenic hormones. This inhibition of beta-casein production occurs at the transcriptional level and in the transforming growth factor-alpha-transformed cells is due to an autocrine mechanism involving the activation of the epidermal growth factor receptor. This suggests that, although the c-erbB-2 and epidermal growth factor receptors are structurally quite similar, their activation has different effects upon mammary epithelial cell differentiation.
Animals↗
Transforming growth factor-alpha expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-ras protooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-alpha complementary DNA leads to transformation.
MCF-10A cells are a spontaneously immortalized normal human mammary epithelial cell line. MCF-10A cells were transfected with two expression vector plasmids containing either a human point-mutated c-Ha-ras protooncogene or the rat c-neu protooncogene. c-Ha-ras-transfected MCF-10A cells grow as colonies in soft agar, exhibit a 3- to 4-fold increase in their growth rate in serum-free medium, and show a reduced mitogenic response to exogenous epidermal growth factor (EGF) or transforming growth factor-alpha (TGF alpha) as compared to MCF-10A cells. c-Ha-ras-transfected MCF-10A cells express a 4- to 8-fold increase in TGF alpha mRNA levels and secrete 4- to 6-fold more TGF alpha protein as compared to MCF-10A cells. Addition of either an anti-TGF alpha neutralizing monoclonal antibody or an anti-EGF receptor blocking monoclonal antibody to the Ha-ras-transformed MCF-10A cells produces a 50 to 80% inhibition of colony formation of these cells in soft agar. c-neu-transfected MCF-10A cells grown in soft agar and exhibit an increase in their growth rate in serum-free medium at a level comparable to that observed in Ha-ras-transformed MCF-10A cells. Addition of an anti-c-erbB-2 monoclonal antibody inhibits the anchorage-independent growth of these cells in soft agar. However, c-neu-transformed MCF-10A cells show no increase in TGF alpha secretion and no change in their responsiveness to exogenous EGF or TGF alpha. A recombinant retroviral vector containing the human TGF alpha gene was also introduced into MCF-10A cells. TGF alpha-infected MCF-10A cells secrete 15- to 20-fold more TGF alpha protein than MCF-10A cells, form colonies in soft agar, exhibit an enhanced growth rate in serum-free medium, and show a decreased mitogenic response to exogenous EGF or TGF alpha at a level equivalent to Ha-ras-transformed MCF-10A cells. Growth of TGF alpha-infected MCF-10A cells in soft agar is completely inhibited by anti-TGF alpha neutralizing or anti-EGF receptor blocking monoclonal antibodies. These results suggest that TGF alpha is an intermediary in the transformation of human mammary epithelial cells by an activated c-Ha-ras gene, but not by the c-neu gene, and demonstrate that overexpression of this growth factor is able to transform immortalized human mammary epithelial cells which also express a sufficient complement of functional EGF receptors.
Blotting, Northern↗
Transforming growth factor-alpha: an oncodevelopmental growth factor.
Transforming growth factor-alpha (TGF-alpha) is a 50-amino-acid mitogenic peptide that is structurally and, in some cases, functionally related to members of the epidermal growth factor (EGF) family of peptides. TGF-alpha is initially synthesized as a high-molecular-weight, glycosylated, membrane-associated precursor of approximately 160 amino acids. The low-molecular-weight TGF-alpha peptide as well as the precursor are biologically active in a number of systems and can function as transforming proteins when overexpressed. TGF-alpha binds to and activates the EGF receptor, and TGF-alpha and the EGF receptor are coexpressed in a number of human and rodent tumors and tumor cell lines--which suggests that TGF-alpha can function as an autocrine or paracrine growth factor. TGF-alpha is transiently expressed in some fetal and adjacent maternal tissues during development and is also expressed in a number of adult tissues; this pattern of expression suggests that the growth factor is involved in several distinct physiological functions.
Amino Acid Sequence↗
Isolation of a new class of 'flat' revertants from ras-transformed NIH3T3 cells using cis-4-hydroxy-L-proline.
A new class of nontransformed revertant cells has been isolated from the ras-transformed cell line DT using cis-4-hydroxy-L-proline (CHP) as a selective agent. The new revertants, CHP 9CJ and CHP CB4, each contain two copies of the v-Ki-ras gene, elevated levels of phosphorylated p21ras protein, and rescuable transforming virus, indicating that the revertant phenotype observed in these cells does not result from inactivation of v-Ki-ras or inhibition of its expression. Both CHP 9CJ and CHP CB4 revertants show a greatly reduced ability to form colonies in soft agar and to produce tumors in syngeneic mice. CHP 9CJ cells are resistant to retransformation by ras and by additional oncogenes that do not encode tyrosine kinases. A comparison of oncogene resistance patterns in these CHP-derived revertants with those from our original ouabain-derived revertants fos C-11 and F-2 indicates that oncogenes may be divided into four general groups. Oncogenes that encode proteins structurally related to p21ras comprise the first group. The second group contains only tyrosine kinase-encoding oncogenes. The third group is composed of 'nuclear', e.g. fos, and 'cytoplasmic' serine-threonine-encoding oncogenes such as mos and raf. The fourth group contains the oncogenes sis and fms.
Animals↗
The mouse int-2 gene exhibits basic fibroblast growth factor activity in a basic fibroblast growth factor-responsive cell line.
The int-2 protein is related to basic fibroblast growth factor (bFGF) by amino acid sequence homology. To assess its biological activity, we constructed retroviral vectors containing four variants of mouse int-2 complementary DNA under the transcriptional control of the beta-actin promoter and tested their effects on human SW13 adrenal cortical tumor cells. This cell line specifically requires bFGF, interleukin 1, or transforming growth factor e for anchorage-independent growth in soft agar. Despite encoding a signal sequence that should direct the protein to the secretory pathway, vectors containing unmodified int-2 complementary DNA, or a form optimized for translation initiation at the AUG codon, were incapable of inducing SW13 growth in soft agar. However, SW13 transfectants expressing a construct (pSP1), in which a mouse immunoglobulin signal peptide sequence is linked to the int-2 coding sequences, grew well in soft agar. The concentrated conditioned medium from these pSP1-transfected cells supported anchorage-independent growth of SW13 indicator cells and competed with bFGF for binding to receptors. Western blot analysis with an int-2-specific antiserum detected Mr 30,000-32,000 int-2 products in cell extracts and conditioned medium from pSP1-transfected clones, whereas the conditioned medium from these and other SW13 clones contained only low levels of bFGF as measured in a specific radioimmunoassay. These data suggest that the product of the int-2 gene can functionally replace bFGF in modulating the anchorage-independent growth of SW13 cells.
Amino Acid Sequence↗
mRNA expression of transforming growth factor alpha in human breast carcinomas and its activity in effusions of breast cancer patients.
Transforming growth factor alpha (TGF alpha) mRNA expression was measured by Northern blot analysis in 18 human, primary, infiltrating, ductal breast carcinomas. Expression of a 4.8-kilobase TGF alpha mRNA transcript was detected in nine of 18 tumors. No evidence was observed of any gross amplifications or major rearrangements of the TGF alpha gene in the breast carcinoma specimens. Biologically active and immunoreactive TGF alpha was measured in the pleural effusions or in the ascitic fluids from 37 noncancer and 63 cancer patients. The TGF alpha activity detected ranged from 0.2 to 26 ng/mL in most effusions from both groups. However, 29 of 63 (46%) of the effusions from cancer patients exhibited TGF alpha levels that were 6 ng/mL or higher, whereas only seven of 37 (19%) of those from noncancer patients exceeded this level (P less than .03). In particular, effusions obtained from breast cancer patients showed a significantly higher level of TGF alpha, compared with those from noncancer patients (P less than .001). Effusions from 14 cancer patients also contained elevated levels of two tumor-associated antigens, CEA and/or TAG-72, and within this group, nine also had elevated levels of TGF alpha.
Ascitic Fluid↗
Transformation of mouse mammary epithelial cells with the Ha-ras but not with the neu oncogene results in a gene dosage-dependent increase in transforming growth factor-alpha production.
An enhanced expression of transforming growth factor-alpha (TGF alpha) was demonstrated in two clones of NOG-8 mouse mammary epithelial cells, NOG-8 SR1 and NOG-8 SR2, that have been transformed by a v-Ha-ras oncogene. The amount of TGF alpha production in NOG-8 SR1 and NOG-8 SR2 cells was dependent on the level of p21ras expression in these clones, which directly correlated with their cloning efficiency in soft agar. There was also a decrease in the number of epidermal growth factor (EGF) receptors on the NOG-8 SR1 and NOG-8 SR2 cells that is proportional to the amount of TGF alpha secreted. These effects were specific for ras because neu-transformed NOG-8 cells grew in soft agar at a comparable level to NOG-8 SR2 cells yet did not show any increase in TGF alpha production or change in EGF receptor expression.
Animals↗
Site-selective 8-chloroadenosine 3',5'-cyclic monophosphate inhibits transformation and transforming growth factor alpha production in Ki-ras-transformed rat fibroblasts.
A site-selective cAMP analog, 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP), was demonstrated to be a potent inhibitor of both the monolayer and soft agar growth of normal rat kidney (NRK) fibroblasts that had been transformed with the v-Ki-ras oncogene or treated with transforming growth factor alpha (TGF alpha). The growth inhibition was dose dependent and reversible and was accompanied by reversion of the transformed phenotype, suppression of TGF alpha production, and a decrease in p21 ras protein levels. These effects of 8-Cl-cAMP were linked to the cAMP analog's selective modulation of the type I and type II cAMP-dependent protein kinase regulatory subunits, RI and RII, present in Ki-ras-transformed and TGF alpha-treated NRK cells.
8-Bromo Cyclic Adenosine Monophosphate↗