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Glutaraldehyde-fixed transformed and non-transformed cells induce contact-dependent inhibition of growth in non-transformed C3H/10T1/2 mouse fibroblasts, but not in 3-methylcholanthrene-transformed cells.

C3H/10T1/2 mouse fibroblasts showed a pronounced inhibition of growth when reaching a critical cell density. The situation of high cell density could be mimicked by the addition of glutaraldehyde-fixed cells to sparsely seeded proliferating cells. Treatment of the C3H/10T1/2 cells with 3-methylcholanthrene led to a high frequency of piled up foci (118 type II and type III foci in 78 cultures). Cells of a type III focus of a treated culture were cloned. These cells grew in soft-agar and reached 10 times higher cell densities when grown in culture dishes, than did their non-transformed counterparts. Glutaraldehyde-fixed transformed cells did not differ from fixed non-transformed cells in the ability to inhibit the growth of sparsely seeded non-transformed cells. On the other hand, both the addition of fixed normal or transformed C3H/10T1/2 cells did not affect the growth rate of transformed cells. In a concept explaining the density-dependent inhibition of growth of non-transformed cells by a specific interaction of plasma membrane-localized effectors with plasma membrane-localized receptors, the present findings would indicate that the transformed cells used express active effectors but are functionally defective in the receptors or in the signal transmission.

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-beta1 induces transforming growth factor-beta1 and transforming growth factor-beta receptor messenger RNAs and reduces complement C1qB messenger RNA in rat brain microglia.

Transforming growth factor-beta1 is a multifunctional peptide with increased expression during Alzheimer's disease and other neurodegenerative conditions which involve inflammatory mechanisms. We examined the autoregulation of transforming growth factor-beta1 and transforming growth factor-beta receptors and the effects of transforming growth factor-beta1 on complement C1q in brains of adult Fischer 344 male rats and in primary glial cultures. Perforant path transection by entorhinal cortex lesioning was used as a model for the hippocampal deafferentation of Alzheimer's disease. In the hippocampus ipsilateral to the lesion, transforming growth factor-beta1 peptide was increased >100-fold; the messenger RNAs encoding transforming growth factor-beta1, transforming growth factor-beta type I and type II receptors were also increased, but to a smaller degree. In this acute lesion paradigm, microglia are the main cell type containing transforming growth factor-beta1, transforming growth factor-beta type I and II receptor messenger RNAs, shown by immunocytochemistry in combination with in situ hybridization. Autoregulation of the transforming growth factor-beta1 system was examined by intraventricular infusion of transforming growth factor-beta1 peptide, which increased hippocampal transforming growth factor-beta1 messenger RNA levels in a dose-dependent fashion. Similarly, transforming growth factor-beta1 increased levels of transforming growth factor-beta1 messenger RNA and transforming growth factor-beta type II receptor messenger RNA (IC(50), 5pM) and increased release of transforming growth factor-beta1 peptide from primary microglia cultures. Interactions of transforming growth factor-beta1 with complement system gene expression are also indicated, because transforming growth factor-beta1 decreased C1qB messenger RNA in the cortex and hippocampus, after intraventricular infusion, and in cultured glia. These indications of autocrine regulation of transforming growth factor-beta1 in the rodent brain support a major role of microglia in neural activities of transforming growth factor-beta1 and give a new link between transforming growth factor-beta1 and the complement system. The auto-induction of the transforming growth factor-beta1 system has implications for transgenic mice that overexpress transforming growth factor-beta1 in brain cells and for its potential role in amyloidogenesis.

Alzheimer Disease↗

Transformation of rodent fibroblasts by herpes simplex virus: presence of morphological transforming region 1 (MTR 1) is not required for the maintenance of the transformed state.

Studies on the mechanisms of transformation of mammalian cells by herpes simplex virus (HSV) in vitro have been prevented so far by the extremely low transformation frequencies obtained in monolayer culture. Here we present a transformation system that relies on the direct seeding in soft agar of infected single cells, thus avoiding negative interactions between normal and transformed cells. We took advantage of HSV-I temperature-sensitive mutants at the UL9 locus, which codes for a DNA-binding protein necessary for viral DNA replication. At the non-permissive temperature, viral DNA synthesis and late gene expression are prevented. Viral gene expression is restricted to immediate early and early genes. Induction of transformation was highly efficient in our one-step transformation system. It depended on intact viral particles and viral DNA. Immediate early and/or early viral gene expression was sufficient to induce transformation. Colonies were stably transformed and did not show any rescue of viable virus after temperature downshift and co-cultivation with susceptible cells. Transformed cells maintained the transformed state in the absence of viral DNA. Our data therefore support the "hit-and-run" hypothesis for the transforming effect of HSV.

Animals↗

P53 transformation-related protein accumulates in the nucleus of transformed fibroblasts in association with the chromatin and is found in the cytoplasm of non-transformed fibroblasts.

The subcellular localization of the p53 molecule was studied in transformed and non-transformed fibroblasts. A newly established transformed cell line obtained by treating primary embryonic mouse cells in vitro with the chemical carcinogen methylcholanthrene was compared with the embryonic parent fibroblasts. The transformed cells lost the spindle shape characteristic of the parent fibroblasts, acquired an accelerated growth rate, developed into tumors when injected into syngeneic mice and expressed high levels of p53 synthesis estimated by immunoprecipitation of [35S]methionine-labeled cell extracts. The cellular localization of the p53 molecule was studied by immunofluorescent staining of fixed cells with monoclonal antibodies and by immunoprecipitation of [35S]-methionine-labeled p53 from various subcellular fractions. p53 was mainly found in the nucleus of the transformed fibroblast, while in the parent non-transformed primary embryonic cells, p53 was detected in the cytoplasm in a Triton X-100 soluble fraction, and associated with the cytoskeleton. The modulated distribution of p53 was also confirmed by analyzing a wide range of independently established transformed and non-transformed fibroblastic cell lines growing in vitro. The switch from the cytoplasmic localization of p53 in the non-transformed fibroblasts to a chromatin-associated accumulation in the transformed cells suggests a possible mechanism by which this protein may function in the transformed fibroblasts.

Animals↗

Kirsten murine sarcoma virus transformed cell lines and a spontaneously transformed rat cell-line produce transforming factors.

We have examined culture fluids from a variety of Kirsten murine sarcoma virus (KiMSV) transformed rat and mouse cells for the presence of factors which induce normal Rat-1 cells to assume the transformed phenotype. All KiMSV transformants produced transforming factor (TF). Revertants of KiMSV transformed rat or mouse failed to release TF as did normal rat or mouse cells. Cells transformed by a temperature sensitive mutant of KiMSV produced TF at the permissive temperature but not at the nonpermissive temperature. Further, cells from a spontaneous transformant of Rat-1 cells also produced TF. TF is a small polypeptide which competes for the epidermal growth factor receptor. Its effect upon normal cells is reversible and requires de novo RNA and protein synthesis. Cells treated with TF lose the actin fibers observed in normal fibroblasts, assume a transformed cell morphology, become anchorage independent for growth, grow in low concentrations of serum, grow to a high cell density, and have an increased rate of hexose uptake.

Actins↗

Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: effect of transformation technique on the transformed phenotype.

Simian virus 40 tsA-transformed BALB/c-3T3 cells isolated as foci of overgrowth in liquid medium were compared with those isolated as colonies in soft agar. Efficiencies of transformation were equivalent in the two procedures. Cells isolated as foci were able to grow in agar and vice versa. No difference in temperature sensitivity of the transformed phenotype was detected when tsA transformants selected in agar were compared with those selected as foci. The use of the two different transformation procedures, then, did not form the basis for generation of different transformed phenotypes, and transformants generated in both ways were dependent upon expression of the A gene for maintenance of the transformed state.

Agar↗

Radiation-induced cell transformation: transformation efficiencies of different types of ionizing radiation and molecular changes in radiation transformants and tumor cell lines.

This study aims to compare the efficiencies of 5.4 keV soft X-rays, alpha-particles, and gamma-rays in transforming C3H 10T1/2 cells and to assess the sequence of cellular and molecular changes during the process of radiation-induced transformation of Syrian hamster embryo (SHE) cells. The somewhat more densely ionizing soft X-rays are more effective than gamma-rays both for cell inactivation and cell transformation. The relative biological effectiveness (RBE) appears to be independent of dose; it is approximately 1.3 for either end point. The RBE of alpha-particles versus gamma-rays, on the other hand, increases with decreasing dose; the dose dependence is somewhat more apparent for cell transformation than for cell inactivation. SHE cells transformed by different types of ionizing radiation and related tumor cell lines isolated from nude mice tumors were found to have a distinct growth advantage compared to primary SHE cells, documented by higher plating efficiencies, shorter doubling times, and higher cloning efficiencies in semisolid medium. Most transformed and tumor cell lines that were investigated have elevated mRNA levels for the H-ras gene, some of them show restriction fragment length polymorphisms of the H-ras gene, and some exhibit a substantially amplified c-myc gene. In a sequence analysis of the Syrian hamster H-ras gene of eight tumor cell lines from radiation transformants, we have not found any mutation in codons 12, 13, 59, 61, nor in the flanking regions of these codons. The transformed and tumor cell lines tend to have lower chromosome numbers than primary SHE cells.

Alpha Particles↗

Integration efficiency in DNA-induced transformation of Pneumococcus. I. A method of transformation in solid medium and its use for isolation of transformation-deficient and recombination-modified mutants.

A method of transformation on solid medium especially adapted for pneumococcus has been developed. Under specific conditions, all colonies that are allowed to grow in the presence of transforming DNA for six hours give rise to transformed bacteria. Combined with replica plating this technique has been used to isolate mutants modified with regard to recombination. Most of the mutants found are transformation-defective and show a large diversity in their response to ultraviolet light. Some of these mutants have lost their ability to take up transforming DNA. One shows a reduced yield of transformants for a given quantity of DNA taken up. Mutants that manifest altered behavior with regard to marker efficiencies have also been isolated. One of these exhibits a decrease in the transformation efficiency of only the high efficiency markers and two mutants show a decrease in the transformation efficiency of the low efficiency markers.

Aminopterin↗

Induction of foci of morphologically transformed cells in synchronized populations of 10T1/2 cells by N-methyl-N'-nitro-N-nitrosoguanidine and the effect of spontaneous transformation on calculated transformation frequency.

Exposure of synchronized C3H10T1/2 (clone 8) cell populations of various sizes to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at a concentration of 2 micrograms/ml for 30 min at 24 h after release from confluence-induced arrest of proliferation produced neoplastic transformation (formation of foci of morphologically altered cells) by a random but episodic process in a small fraction of the cells at risk soon after treatment. The fraction of dishes that contained type II or type III foci increased as the number of cells at risk increased. In contrast, the development of spontaneous foci is a stochastic process that depends on the number of new cells that form during population growth and is independent of the number of cells that are plated (J. W. Grisham et al., Cancer Res., 48: 5969-5976,1988). When there were small numbers of cells at risk, spontaneous formation of foci was a source of considerable error in evaluating MNNG-induced transformation frequency. In surviving cell populations of less than 1000-3000 cells/100-mm dish, the frequency of induction of foci by MNNG could not be distinguished statistically from the frequency with which foci were expected to form spontaneously. When the fraction of MNNG-treated dishes that contained foci was adjusted for the fraction of pooled control dishes that contained foci, the number of foci induced by a uniform dose of MNNG was found to vary with the number of surviving cells. However, the MNNG-induced transformation frequencies calculated by the Poisson method were independent of the size of the population of cells at risk, provided the population of cells at risk was of sufficient size to allow spontaneous and induced transformation to be distinguished statistically. The results of this study show that the frequency of MNNG-induced transformation can be quantitated in cultures of 10T1/2 cells that contain varying but sufficient numbers of cells at risk when spontaneous transformation is considered. Furthermore, these observations suggest that MNNG-induced transformation of 10T1/2 cells occurs with the frequency and characteristics of a mutation-like change involving a single gene.

Animals↗

Transforming growth factor beta 1 partially suppresses the transformed phenotype of ras-transformed hepatocytes.

The effect of transforming growth factor beta type 1 (TGF-beta 1) on DNA synthesis, anchorage-dependent and anchorage-independent proliferation, cytoskeletal organization, and gene expression in ras-transformed simian virus 40 (SV40)-immortalized hepatocyte cell lines was measured. An SV40-immortalized cell line (CWSV1), a control neo-transfected and selected cell line (N1), and neo+ras-transfected and selected cell lines (NR3 and NR4) were used for this study. CWSV1 and N1 cells do not grow in soft agarose and are not tumorigenic. The ras-transformed hepatocytes NR3 and NR4 grow in soft agar and are tumorigenic. TGF-beta 1 treatment did not inhibit DNA synthesis or anchorage-dependent growth in the SV40-immortalized hepatocyte cell line CWSV1 or in the ras-transformed hepatocytes. TGF-beta 1 treatment inhibited anchorage-independent growth, increased actin cytoskeleton organization, and altered the morphology of ras-transformed hepatocytes; that is, with regard to all three of these properties, TGF-beta 1-treated ras-transformed hepatocytes more closely resembled the immortalized parent cell line. c-Ha-ras and c-myc RNA levels were not altered in TGF-beta 1-treated NR4 cells. TGF-beta 1 treatment did alter expression of some genes in NR4 cells. The level of expression of alpha 1 integrin RNA was higher in CWSV1 cells than in NR4 cells and increased in NR4 cells when they were treated with TGF-beta 1. Similarly, the levels and profiles of integrins on the cell surface of CWSV1 cells compared to NR4 cells, as determined by cell surface protein iodination, differed and in TGF-beta 1-treated NR4 cells more closely resembled the surface integrin profile for CWSV1 cells.

Actins↗

Growth control in simian virus 40-transformed rat cells: temperature-independent expression of the transformed phenotype in tsA transformants derived by agar selection.

Fisher rat fibroblasts (FR 3T3), transformed with the tsA30 mutant of simian virus 40 and selected by colony formation in soft agar, maintained the transformed phenotype at high temperature, whereas most transformants isolated from foci were found to undergo a phenotypic reversion toward the normal state in their saturation density, ability to grow in soft agar, and rate of 2-deoxyglucose transport. The temperature-independent phenotype observed in agar-selected transformants was not due to a reversion of the viral mutation. These results, similar to those previously obtained with polyoma virus tsa mutants, further suggest that two distinct mechanisms may operate in both cases for maintaining the transformed phenotype. Immunofluorescence studies suggested a different regulation of T antigen synthesis in these two classes of transformants.

Agar↗

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↗

[Genetic transformation of somatic cells. IV. The rate of loss of transformant phenotype depends on the structure of the transforming DNA and changes when the cells are treated with the tumor promotor 12-o-tetradecanoyl-phorbol-13-acetate].

Analysis was made of the phenotype stability of some clones of thymidine kinase deficient (TK-) Chinese hamster cells transformed by thymidine kinase gene (TK-gene) of Herpes simplex virus type (HSV 1). The presence of a fragment of human satellite DNA III in the plasmid DNA carrying the TK-gene of HSV 1 reduced notably the rate of the loss of TK+-phenotype, and the treatment of the cells with a tumour promoter--12-o-tetradecanoyl-phorbol-13-acetate--immediately after transformation destabilizes TK+-phenotype of transformant clones. Removal of the eukaryotic carrier DNA for the plasmid DNA without the TK-gene of HSV 1 destabilizes the clone transformant phenotype. Changes in the structure of the plasmid DNA containing no TK-gene of HSV 1 and introduced into cells simultaneously with TK-gene containing plasmids affects the rate of the loss of TK+-phenotype transformed cells.

Animals↗

Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences.

When the yeast Saccharomyces cerevisiae was transformed with DNA that shares no homology to the genome, three classes of transformants were obtained. In the most common class, the DNA was inserted as the result of a reaction that appears to require base pairing between the target sequence and the terminal few base pairs of the transforming DNA fragment. In the second class, no such homology was detected, and the transforming DNA was integrated next to a CTT or GTT in the target; it is likely that these integration events were mediated by topoisomerase I. The final class involved the in vivo ligation of transforming DNA with nucleus-localized linear fragments of mitochondrial DNA.

Base Sequence↗

Retinoic acid and calcium regulation of p53, transforming growth factor-beta 1, and transforming growth factor-alpha gene expression and growth in adenovirus 12-SV40-transformed human tracheal gland epithelial cells.

Vitamin A and calcium are important regulators of growth and differentiation of epithelial cells and are intimately involved in preneoplastic and neoplastic transformation. It has been proposed that their effects are mediated by autocrine/paracrine positive and negative regulators of growth. The objectives of this investigation were to examine the effects of all-trans retinoic acid (RA) and Ca2+ on cell proliferation, anchorage-independent growth (AIG), and on the expression of transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta 1 (TGF-beta 1), and p53 tumor suppressor genes in human tracheal gland epithelial (HTGE) cells immortalized by adenovirus 12-simian virus 40 (Ad12-SV40) hybrid. Cells exhibiting the transformed phenotype, AIG, were maintained in serum-free culture conditions. Calcium effects were examined at 0.15, 0.50, 1.0, and 2.0 mM concentrations. The effects of RA were determined with 10(-9), 10(-7), and 10(-6) M concentrations. Gene expression was examined by Northern and Western analyses. Ca2+ had no significant effect on cell proliferation, but it enhanced the expression of TGF-beta 1 gene and slightly inhibited p53 expression. Ca2+ had no effect on TGF-alpha. RA inhibited both cell proliferation and AIG growth, which was accompanied by enhanced expression of p53. RA had no significant effect on the expression of TGF-alpha and TGF-beta 1 genes. These results demonstrate that RA regulates growth of HTGE cells mainly by upregulating the p53 gene; Ca2+, which enhances TGF-beta 1 expression, had no effect on growth.

Adenoviruses, Human↗

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↗

Epidermal growth factor-like transforming growth factor. I. Isolation, chemical characterization, and potentiation by other transforming factors from feline sarcoma virus-transformed rat cells.

An acid-stable transforming growth factor (TGF) that interacts with epidermal growth factor (EGF) receptors and is structurally related to EGF was isolated from serum-free culture fluids of Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells. Purification of this EGF-like TGF (eTGF) was achieved by molecular filtration chromatography and successive reverse-phase high pressure liquid chromatography steps on octadecyl support eluted with acetonitrile and 1-propanol gradients, respectively. Rat eTGF consists of a 7.4-kD single polypeptide chain that co-migrates with biological activity in dodecyl sulfate-polyacrylamide electrophoresis gels. Like preparations of a related TGF from human melanoma cells (Marquardt, H., and Todaro, G.J. (1982) J. Biol. Chem. 257, 5220-5225), but unlike EGF from rat, human, or mouse, rat eTGF has phenylalanine and lacks methionine. However, the sequence of the first 30 amino acid residues in rat eTGF is H2N-Val-Val-Ser-His-Phe-Asn-Lys-Cys-Pro-Asp-Ser-His-Thr-Gln-Tyr-Cys-Phe-His-Gly - Thr-(x)-Arg-Phe-Leu-Val-Gln-Glu-Glu-(Lys)-(Lys)-, which is significantly (20% and 28%) homologous to the NH2-terminal region of mouse EGF and human EGF, respectively. In addition to eTGF, molecular filtration chromatography of acid-soluble extracts from medium conditioned by FeSV-Fre cells resolved a 14-kD transforming factor(s) apparently devoid of intrinsic mitogenic activity but able to elicit a strong anchorage-independent growth response in the presence of eTGF or EGF. These results show that: 1) a 7.4-kDa TGF structurally and functionally related to EGF has been isolated from FeSV-Fre cells and 2) the full anchorage-independent growth-promoting activity of medium conditioned by FeSV-Fre cells is due to the coordinate action of at least two types of factors, the 7.4-kDa eTGF and a second 14-kDa transforming factor(s).

Amino Acid Sequence↗