pp60v-src transformation of rat cells but not chicken cells strongly correlates with low-affinity phosphopeptide binding by the SH2 domain.

Substrates critical for transformation by pp60v-src remain unknown, as does the precise role of the src homology 2 (SH2) domain in this process. To continue exploring the role of the SH2 domain in pp60v-src-mediated transformation, site-directed mutagenesis was used to create mutant v-src alleles predicted to encode proteins with overall structural integrity intact but with reduced ability to bind phosphotyrosine-containing peptides. Arginine-175, which makes critical contacts in the phosphotyrosine-binding pocket, was mutated to lysine or alanine. Unexpectedly, both mutations created v-src alleles that transform chicken cells with wild-type (wt) efficiency and are reduced for transformation of rat cells; these alleles are host dependent for transformation. Additionally, these alleles resulted in a round morphological transformation of chicken cells, unlike 12 of the 13 known host-dependent src SH2 mutations that result in a fusiform morphology. Analysis of phosphopeptide binding by the mutant SH2 domains reveal that the in vitro ability to bind phosphopeptides known to have a high affinity for wt src SH2 correlates with wt (round) morphological transformation in chicken cells and in vitro ability to bind phosphopeptides known to have a low affinity for wt src SH2 correlates with rat cell transformation. These results suggest that the search for critical substrates in rat cells should be among proteins that interact with pp60v-src with low affinity.

Transformation and pp60v-src autophosphorylation correlate with SHC-GRB2 complex formation in rat and chicken cells expressing host-range and kinase-active, transformation-defective alleles of v-src.

The biochemical properties of several pp60v-src substrates believed to participate in src-mediated transformation were examined in cells expressing a kinase-active, transformation-defective v-src allele (v-src-F172 delta/Y416F) and its parental allele, v-src-F172 delta, a host-range--dependent allele that transforms chicken cells to a fusiform morphology, but does not transform rat cells. Because pp60v-src-F172 delta is dependent on autophosphorylation for transforming ability, these alleles provide a unique opportunity to examine the role of pp60v-src autophosphorylation in regulating substrate interactions. Increased pp125FAK tyrosine phosphorylation and high levels of pp60v-src-associated phosphotidylinositol-3' kinase activity were detected specifically in chicken cells exhibiting round, refractile transformation but not in cells transformed to a fusiform morphology. Increased pp125FAK kinase activity, but not increased pp125FAK tyrosine-phosphorylation correlated with pp60v-src autophosphorylation and increased anchorage-independent growth. Thus, pp125FAK and PI3'K may participate in morphological transformation by v-src. Furthermore, association of phosphorylated SHC with the adapter GRB2 correlated with increased anchorage-independent growth (and autophosphorylation) in both rat and chicken cells independent of the morphological phenotype induced. Therefore, host-range dependence for transformation may be regulated through association of SHC with GRB2, thus implicating SHC as a crucial substrate for src-dependent transformation.

Functional evidence for a novel human breast carcinoma metastasis suppressor, BRMS1, encoded at chromosome 11q13.

We previously showed that introduction of a normal, neomycin-tagged human chromosome 11 reduces the metastatic capacity of MDA-MB-435 (435) human breast carcinoma cells by 70-90% without affecting tumorigenicity, suggesting the presence of one or more metastasis suppressor genes encoded on human chromosome 11. To identify the gene(s) responsible, differential display comparing chromosome 11-containing (neo11/ 435) and parental, metastatic cells was done. We describe the isolation and functional characterization of a full-length cDNA for one of the novel genes, designated breast-cancer metastasis suppressor 1 (BRMS1), which maps to human chromosome 11q13.1-q13.2. Stably transfected MDA-MB-435 and MDA-MB-231 breast carcinoma cells still form progressively growing, locally invasive tumors when injected into mammary fat pads but are significantly less metastatic to lungs and regional lymph nodes. These data provide compelling functional evidence that breast-cancer metastasis suppressor 1 is a novel mediator of metastasis suppression in human breast carcinoma.

Highly conserved amino acids in the SH2 and catalytic domains of v-src are altered in naturally occurring, transformation-defective alleles.

We have identified 11 novel point mutations that abolish the transforming capacity of the oncogene v-src. These transformation-defective alleles were originally identified in morphologically flat subclones of rat cells transformed by wild type v-src. Nine of the mutations affect amino acid residues that are highly conserved in the catalytic domain of pp60v-src and completely abolish kinase activity. The other 2 mutations alter conserved residues in the SH2 domain (Phe-172 replaced with Val in one case [F172V] and Leu-186 replaced with Phe in the other [L186F]), drastically reducing, but not eliminating, kinase activity. The enzymatic and transforming functions of one of the SH2 mutants, L186F are host dependent; the mutant protein is active in chicken cells, but inactive in rat cells, as previously observed for some other SH2 mutants. These results are interpreted in relation to the recently described three-dimensional structures of SH2 domains and of the catalytic domain of a protein kinase. In addition, they support a role for the SH2 domain in the regulation of kinase activity.

Identification of a complete Cek7 receptor protein tyrosine kinase coding sequence and cDNAs of alternatively spliced transcripts.

Receptor protein tyrosine kinases (RPTK) are critical components of signal transduction pathways in multicellular organisms. Identification of new RPTK constitutes an initial step in understanding the variety of signalling pathways in which these proteins participate. In this study, a cDNA containing a complete coding sequence for Cek7 (chicken RPTK) has been cloned from a chicken embryo expression library using anti-phosphotyrosine antibodies (Ab). Cek7 is a member of the EPH (human RPTK) subfamily of RPTK; this subfamily is characterized by extracellular domains containing an immunoglobulin-like motif, a Cys-rich region and two fibronectin type-III repeats. Analysis of additional cDNAs revealed that two positions of alternative splicing in primary transcripts may produce several isoforms of this RPTK; cDNAs corresponding to three isoforms of this receptor are reported. These isoforms are predicted to have altered extracellular ligand-binding domains and/or altered cytoplasmic juxtamembrane regions. The nucleotide sequence of cek7 cDNAs identified in this study diverges at the 3' end from the sequence found in a recently described partial cek7 cDNA [Sajjadi and Pasquale, Oncogene 8 (1993) 1807-1813]. Therefore, a third position of alternative splicing may produce Cek7 RPTK with divergent C-terminal tails. RNA blot analysis revealed expression of this receptor at highest levels in the central nervous system and eyes of 10-day-old chicken embryos.

Analysis of mechanisms underlying BRMS1 suppression of metastasis.

Introduction of normal, neomycin-tagged human chromosome 11 (neo11) reduces the metastatic capacity of MDA-MB-435 human breast carcinoma cells by 70-90% without affecting tumorigenicity. Differential display comparing MDA-MB-435 and neo11/435 led to the discovery of a human breast carcinoma metastasis suppressor gene, BRMS1, which maps to chromosome 11q13.1-q13.2. Stable transfectants of MDA-MB-435 and MDA-MB-231 breast carcinoma cells with BRMS1 cDNA still form progressively growing, locally invasive tumors when injected in mammary fat pads of athymic mice but exhibit significantly lower metastatic potential (50-90% inhibition) to lungs and regional lymph nodes. To begin elucidating the mechanism(s) of action, we measured the ability of BRMS1 to perturb individual steps of the metastatic cascade modeled in vitro. Consistent differences were not observed for adhesion to extracellular matrix components (laminin, fibronectin, type IV collagen, type I collagen, Matrigel); growth rates in vitro or in vivo; expression of matrix metalloproteinases, heparanase, or invasion. Likewise. BRMS1 expression did not up regulate expression of other metastasis suppressors, such as NM23, Kai1, KiSS1 or E-cadherin. Motility of BRMS1 transfectants was modestly inhibited (30-60%) compared to parental and vector-only transfectants. Ability to grow in soft agar was also decreased in MDA-MB-435 cells by 80-89%, but the decrease for MDA-MB-231 was less (13-15% reduction). Also, transfection and re-expression of BRMS1 restored the ability of human breast carcinoma cells to form functional homotypic gap junctions. Collectively, these data suggest that BRMS1 suppresses metastasis of human breast carcinoma by complex, atypical mechanisms.

Antisense oligonucleotides to gastrin inhibit growth of human pancreatic cancer.

Human pancreatic cancer is stimulated by the autocrine production of gastrin. In this study, the effects of administration of antisense oligonucleotides to gastrin on growth of pancreatic cancer were evaluated in vitro and in vivo. Log phase BxPC-3 human pancreatic cancer cells in culture were exposed to increasing concentrations (0.5-10 microM) of a synthetic 20-mer antisense phosphorothioate oligonucleotide to gastrin for 48 h and growth was assessed by the cellular proliferation assay. Growth was inhibited up to 88% by anti-gastrin oligonucleotides in a dose-related fashion compared to cells treated with diluent or a randomized sequence with the same composition as the anti-gastrin oligonucleotide. In vivo nude mice bearing BxPC-3 xenografts were treated daily for 14 days with a 0.1-ml intratumoral injection of either anti-gastrin (5 microM), the scrambled sequence control phosphorothioate oligonucleotide (5 microM), or buffer. Tumors from the anti-gastrin-treated mice were significantly smaller in volume and weight and had less gastrin detected by radioimmunoassay than either controls. These results support the role of gastrin as a stimulatory peptide for growth of human pancreatic cancer. Antisense oligonucleotide to gastrin may have a role in the future treatment of patients with pancreatic cancer.

Opioid growth factor regulates the cell cycle of human neoplasias.

The native opioid growth factor (OGF), [Met5]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and migration, as well as tissue organization, during development, cancer, homeostatic cellular renewal, wound healing, and angiogenesis. OGF action is mediated by the OGF receptor (OGFr). To investigate the target of OGF as to cell proliferation, the effects of excess OGF, and a deprivation of OGF-OGFr interaction by an opioid antagonist, naltrexone (NTX), were examined in 3 human cancer cell lines: pancreatic (BxPC-3), colon (HT-29), and head and neck (CAL-27). OGF exposure decreased growth, DNA synthesis, and mitosis, and increased the doubling time from control levels. FACS analysis revealed a marked increase in cells in the G0/G1 phase and compensatory reduction in cells in S and G2/M phases. Consistent with this observation, the percentage of labeled mitosis (PLM) analysis showed a notable increase in the time of the G0/G1 phase. Receptor blockade with NTX increased the rate of growth, length of DNA synthesis and mitotic phases, and decreased doubling time from control values. FACS analysis indicated an increase in the proportion of cells in S and G2/M phases, and a decrease in the number of cells in the G0/G1 phase. PLM evaluation demonstrated a shortening of the length of the S and G2 phases in the 3 cell lines, and decreases in the M and G0/G1 phases in some cancers. These results indicate that OGF action is directed at the G0/G1 phase, but interruption of OGF-OGFr interfacing has widespread repercussions on the cell cycle. The data on blockade of OGF-OGFr during log phase growth suggest a requisite escorting of the growth peptide and its receptor through the cell cycle.

Molecular characterization and distribution of the opioid growth factor receptor (OGFr) in mouse.

The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. The receptor for OGF, OGFr, has been cloned and sequenced in humans and rats. Using primers based on the rat OGFr cDNA, and a mouse embryo expressed sequence tag, the full-length 2.1 kb mouse OGFr cDNA was sequenced. The open reading frame was found to encode a protein of 634 amino acids, and 14 imperfect repeats of 9 amino acids each were a prominent feature. The molecular weight of OGFr was calculated as 70679, and the isoelectric point was 4.5. Northern blot analysis revealed a 2.1 kb OGFr mRNA transcript in adult mouse brain, heart, lung, liver, kidney, and triceps surae muscle. The amino acids for mouse and rat OGFr were 93% similar and 91% identical, but the mouse and human shared only a 70% similarity and a 58% identity. These results emphasize the molecular validity of OGFr, and explain the interaction of OGF with respect to normal and abnormal growth in mouse cells and tissues.

Genotoxic metabolites of estradiol in breast: potential mechanism of estradiol induced carcinogenesis.

Long term exposure to estradiol increases the risk of breast cancer in a variety of animal species, as well as in women. The mechanisms responsible for this effect have not been firmly established. The prevailing theory proposes that estrogens increase the rate of cell proliferation by stimulating estrogen receptor-mediated transcription and thereby the number of errors occurring during DNA replication. An alternative hypothesis proposes that estradiol can be metabolized to quinone derivatives which can react with DNA and then remove bases from DNA through a process called depurination. Error prone DNA repair then results in point mutations. We postulate that these two processes, increased cell proliferation and genotoxic metabolite formation, act in an additive or synergistic fashion to induce cancer. If correct, aromatase inhibitors would block both processes whereas anti-estrogens would only inhibit receptor-mediated effects. Accordingly, aromatase inhibitors would be more effective in preventing breast cancer than use of anti-estrogens. Our studies initially demonstrated that catechol estrogen (CE) quinone metabolites are formed in MCF-7 human breast cancer cells in culture. Measurement of estrogen metabolites and conjugates involved utilization of an HPLC separation coupled with an electrochemical detector. We then utilized an animal model that allows dissociation of estrogen receptor-mediated function from that of the effects of estradiol metabolites. Wnt-1 transgenic mice harboring a knock-out of ERalpha provides a means of examining the effect of estrogen deprivation in the absence of the ER in animals with a high incidence of breast tumors. ERbeta was shown to be absent in the breast tissue of these animals by RNase protection assay. In the breast tissue of these estrogen receptor alpha knock-out (ERKO)/Wnt-1 transgenic mice, we demonstrated formation of genotoxic estradiol metabolites. The ERKO/Wnt-1 breast extracts contained picomole amounts of the 4-catechol estrogens, but not their methoxy conjugates nor the 2-CE or their methoxy conjugates. The 4-CE conjugates with glutathione or its hydrolytic products (cysteine and N-acetylcysteine) were detected in picomole amounts in both tumors and hyperplastic mammary tissue, demonstrating the formation of CE-3,4-quinones. These results are consistent with the hypothesis that mammary tumor development is primarily initiated by metabolism of estrogens to 4-CE and, then, to CE-3,4-quinones, which may react with DNA to induce oncogenic mutations. The next set of experiments examined the incidence of tumors formed in Wnt-1 transgenic mice bearing wild type ERalpha (ER+/+), the heterozygous combination of genes (ER+/ER-) or ERalpha knock-out (ER-/-). To assess the effect of estrogens in the absence of ER, half of the animals were oophorectomized on day 15 and the other half were sham operated. Castration reduced the incidence of breast tumors in all animal groups and demonstrated the dependence of tumor formation upon estrogens. A trend toward reduction in tumor number (not statistically significant at this interim analysis) occurred in the absence of functional ER since the number of tumors was markedly reduced in ERKO animals which were castrated early in life. In aggregate, our results support the concept that metabolites of estradiol may act in concert with ER mediated mechanisms to induce breast cancer.

Cloning, sequencing, chromosomal location, and function of cDNAs encoding an opioid growth factor receptor (OGFr) in humans.

The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. We have cloned and sequenced cDNAs encoding multiple spliced forms of a human OGF receptor. The open reading frame in the longest cDNA was found to encode a protein of 697 amino acids, and 8 imperfect repeats of 20 amino acids each were a prominent feature. Altogether, five alternatively spliced forms were observed. The cDNA hybridized to mRNA from a variety of normal and neoplastic cells and tissues. Functional studies using antisense oligonucleotides to OGFr demonstrated an enhancement in cell growth. Fluorescent in situ hybridization (FISH) experiments showed the chromosomal location to be 20q13.3. This OGF receptor has no homology to classical opioid receptors. These results provide molecular validity for the interaction of OGF and OGF receptor in the regulation of growth processes in humans.

Cloning, sequencing, expression and function of a cDNA encoding a receptor for the opioid growth factor, [Met(5)]enkephalin.

The native opioid growth factor (OGF), [Met(5)]enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing and angiogenesis. OGF action is mediated by a receptor mechanism. We have cloned and sequenced a 2.1-kilobase (kb) cDNA for a receptor to OGF (OGFr). The open reading frame was found to encode a protein of 580 amino acids, and eight imperfect repeats of nine amino acids each were a prominent feature. The protein encoded by this cDNA exhibited the pharmacological, temporal and spatial characteristics of the OGFr. Functional studies using antisense technology demonstrated an enhancement in cell growth. The molecular organization of the OGFr has no homology to classical opioid receptors. These results provide molecular validity for the interaction of OGF and OGFr in the regulation of growth processes.

Quantitative analysis of gastrin mRNA and peptide in normal and cancerous human pancreas.

Gastrin has been shown to stimulate growth of human pancreatic cancer, and does so in an autocrine fashion. In this study, a relationship between gastrin mRNA, peptide, and gastrin receptors were studied in a variety of human pancreatic tissues. Low levels of gastrin mRNA were detected in normal human pancreas by quantitative reverse transcription polymerase chain reaction, but gastrin peptide was not present using radioimmunoassay. Pancreatic adenocarcinoma cells and tissues had 34- to 530-fold higher gastrin mRNA and peptide levels than normal pancreas. Gastrin mRNA and peptide levels were 8,000- and 15,000-fold, respectively, greater in a pancreatic islet cell gastrinoma tumor than in normal pancreas. In comparison to age-matched controls, fasting gastrin plasma levels were 2-fold higher in patients with pancreatic adenocarcinoma and 131-fold greater in subjects with gastrinomas. Receptor binding assays revealed that pancreatic cancer cells had a binding capacity 200-fold greater than gastrinoma tumors, and 10-fold greater than normal pancreas; no differences in K(d) values were recorded between specimens. In contrast to the normal pancreas and gastrinoma tumor, the aggressive behavior of pancreatic adenocarcinoma may be attributed to the autocrine production of gastrin and to the presence of its growth-related receptor.

Autophosphorylation is required for high kinase activity and efficient transformation ability of proteins encoded by host range alleles of v-src.

pp60v-src is a nonreceptor protein tyrosine kinase that can transform both chicken and rodent fibroblasts. The src homology 2 (SH2) domain of this protein serves a critical role in the regulation of protein tyrosine kinase activity. The host range proteins pp60v-src-L, which contains a deletion of a highly conserved residue (Phe-172) in the SH2 domain, and pp60v-src-PPP, which contains a change from a Leu to a Phe at amino acid 186 in the SH2 domain, transform chicken but not rat cells and have slightly reduced kinase activity measured in vitro. The data presented here show that these altered proteins require autophosphorylation on Tyr-416 for high kinase activity and transforming ability. In the absence of autophosphorylation, there is a further decrease of at least threefold in in vitro kinase activity relative to the phosphorylated host range parental protein, no morphological transformation, a reduction in anchorage independent growth, and no disruption of the actin cytoskeleton. In addition, these SH2 mutations abolish the ability of the SH2 domain to bind a phosphorylated peptide that corresponds to the autophosphorylation site of pp60src. Thus, like mutant alleles of c-src encoding transformation competent proteins, and unlike v-src, transformation by pp60v-src-F172 delta and pp60v-src-L186F is dependent on phosphorylation of Y-416 for high kinase activity and transformation ability. The dependence of transformation on phosphotyrosine is not a reflection of an intramolecular interaction between the autophosphorylation site and the SH2 domains since purified SH2 domains are incapable of binding phosphorylated autophosphorylation site peptides in vitro.

Expression of 100,000-Mr simian virus 40 (SV40) tumor antigen in mouse fibroblasts transfected with replication-defective SV40 genomes.

Simian virus 40 early region mutants which are partially or completely replication defective were tested for their ability to transform postcrisis mouse fibroblasts. All mutants tested were capable of generating anchorage-independent transformants. We have previously reported the presence of a variant tumor antigen of 100,000 Mr (100K protein) generated upon transformation by wild-type simian virus 40 virions which correlates with anchorage-independent growth (Chen et al., Mol. Cell. Biol. 1:994-1006, 1981). In this study, none of the mutants tested produced the 100K variant protein at early (before the fifth) passage. Long-term passage (greater than 20 weeks) permitted the expression of this 100K variant in half of the transformants. Thus the phenotype of these mutants is different from both wild-type simian virus 40 (frequently production of 100K by the third passage, and always by the tenth passage) and the origin-minus class of mutants (no production of 100K at any passage).

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.

Cooperativity between the polyamine pathway and HER-2neu in transformation of human mammary epithelial cells in culture: role of the MAPK pathway.

Our experiments were designed to test the cooperativity between the polyamine pathway and HER-2neu in inducing transformation of human mammary epithelial cells in culture. Using the MCF-10A breast epithelial cell line, we observed that induction of overexpression of ornithine decarboxylase (ODC) (the first rate-limiting enzyme in polyamine biosynthesis) markedly potentiated the anchorage-independent growth stimulating effect of the beta2 isoform of neu differentiating factor (NDF) known to activate HER-2neu in MCF-10A cells. ODC overexpression, on the other hand, did not enhance growth in liquid culture, thus pointing to a specific effect on transformation rather than proliferation. ODC-overexpressing MCF-10A cells exhibited increased MAPK phosphorylation in response to administration of NDF and/or epidermal growth factor (EGF). In contrast, the phosphorylation of the members of the stress-activated protein kinase cascade p38 and SEK were not affected by ODC overexpression. Of note, in the absence of EGF and NDF, ODC overexpression failed to induce both clonogenicity and MAPK activation. These results suggest that increased polyamine biosynthetic activity critically interacts with HER-2neu in promoting human mammary cell transformation in culture and that the MAPK cascade is an important mediator of this interaction. If confirmed in future in vivo studies, our results may identify important new targets for the chemoprevention of human breast cancer.

The membrane-binding domain of the Rous sarcoma virus Gag protein.

The Gag protein of Rous sarcoma virus (RSV) can direct particle assembly and budding at the plasma membrane independently of the other virus-encoded products. A previous deletion analysis has suggested that the first 86 amino acids of RSV Gag constitute a large membrane-binding domain that is absolutely required for these processes. To test this hypothesis, we inserted these residues in place of the N-terminal membrane-binding domain of the pp60v-src, a transforming protein whose biological activity requires plasma membrane localization. The ability of the Src chimera to induce cellular transformation suggests that the RSV sequence indeed contains an independent, functional domain.

94,000- and 100,000-molecular-weight simian virus 40 T-antigens are associated with the nuclear matrix in transformed and revertant mouse cells.

A small fraction of the 94,000-molecular-weight multifunctional large T-antigen of simian virus 40 was associated with the nuclear protein matrix derived from simian virus 40-transformed mouse cells. The interaction between this fraction of T-antigen and the matrix was largely or entirely independent of nuclear DNA. Similar amounts of T-antigen were retained by the nuclei of transformed and revertant cell lines. A 100,000-molecular-weight variant of T-antigen, which has been found to correlate specifically with anchorage-independent growth, was present in the nuclear protein matrix of a transformed cell line. A T-antigen-containing revertant selected for the reacquisition of a high serum requirement and an anchorage requirement for growth retained T-antigen in association with its matrix.