Curcumin inhibits tyrosine kinase activity of p185neu and also depletes p185neu.

Curcumin, a natural compound present in turmeric, possessing both anti-inflammatory and antioxidant effects, has been studied vigorously as a chemopreventative in several cancer models. The erbB2/neu gene-encoded p185neu tyrosine kinase is a potent oncoprotein. Overexpression of p185neu in breast cancer is known to be a poor prognostic factor. We investigated the effect of curcumin on p185neu tyrosine kinase and on the growth of breast cancer cell lines. Curcumin dose-dependently inhibited p185neu autophosphorylation and transphosphorylation in vitro and depleted p185neu protein in vivo. It dissociated the binding of p185neu with GRP94 (glucose-regulated protein), a molecular chaperone, and enhanced the depletion of p185neu. The amount of p185neu protein on the cell membrane was drastically decreased after curcumin treatment. These data demonstrated a new mechanism of the anti-tyrosine kinase activity of curcumin. The growth of several breast cancer cell lines was inhibited; the IC50 ranged from 7 to 18 microM, which, however, did not correlate with the expression level of p185neu. Colony formation in the soft agar assay, a hallmark of the transformation phenotype, was preferentially suppressed in p185neu-overexpressing cell lines by 5 microM curcumin (% of control, basal level versus overexpression: 59.3 versus 16.7%; P < 0.001 by Student's t test). Because curcumin effectively inhibited p185neu tyrosine kinase activity by depleting p185neu and potently suppressed the growth of multiple breast cancer cell lines, its therapeutic potential in advanced breast cancer is worthy of further investigation.

Transfection of activated c-H-rasEJ/pSV2neo or pSV2neo genes into rat mammary cells: rapid stimulation of clonal diversification of spontaneous metastatic and cell-surface properties.

We examined whether the conversion of benign rat mammary cells to metastatic cells by transfection of c-H-rasEJ/pSV2neo or control pSV2neo genes results in rapid stimulation of diversification of cellular phenotypes. Transfection of c-H-rasEJ into twice-cloned, stable (greater than 1 year) rat mammary MTC.4 cells, followed quickly by cell cloning, revealed differences in transfected oncogene copy numbers and expression of p21rasEJ. No correlation between c-H-rasEJ copy numbers or the cellular amounts of p21rasEJ or total p21ras and spontaneous metastatic potentials was found. By subcloning transfected cells as soon as possible after gene transfer, we found some rearrangements and amplifications of c-H-rasEJ and heterogeneous spontaneous metastatic potentials. In addition, the expression of the mammary tumor metastasis-associated cell-surface glycoprotein gp580 on untransfected and transfected MTC.4 cells indicated that the cell populations of higher metastatic potential were also more diverse in their cell-to-cell antigen expression than untransfected or non-metastatic, transfected MTC.4 cells. In contrast, the expression of a putative metastasis-suppressor gene, nm23, was unchanged after transfection and subcloning. Control pSV2neo transfections or calcium phosphate treatment alone also resulted in the generation of cellular heterogeneity, although at an apparently lower frequency than c-H-rasEJ transfections, suggesting that transfection of activated, dominantly acting oncogenes, or in some cases control genes, can result in destabilization of transfected cells, rapid diversification and generation of heterogeneity in growth rate, spontaneous metastatic potential and antigen expression.

Lack of correlation between intercellular junctional communication, p21rasEJ expression, and spontaneous metastatic properties of rat mammary cells after transfection with c-H-rasEJ or neo genes.

The loss of intercellular junctional communication between rat 13762NF mammary carcinoma cells and their spontaneous metastatic potentials from the mammary fat pad show a high degree of correlation. We examined a stable, benign, completely junctionally coupled cell clone (MTC.4) of this system after calcium phosphate-mediated transfection with c-H-rasEJ/pSV2neo and control pSV2neo-containing plasmids. There was a good correlation between the copy numbers of c-H-rasEJ incorporated into MTC.4 cells and their contents of p21rasEJ; however, there was not always a correspondence between spontaneous metastatic potential and copy number of c-H-rasEJ or amount of p21rasEJ. After c-H-rasEJ/pSV2neo transfection, some MTC.4 cells lost intercellular junctional communication and became spontaneously metastatic, although some nonmetastatic transfectants also had low percentages of junctionally coupled cells. One of the control pSV2neo transfectants also became metastatic and lost intercellular junctional coupling, and calcium phosphate treatment itself resulted in increased growth rates at mammary fat pad sites and a marginal increase in incidence of spontaneous metastases, both of which preceded loss of intercellular junctional coupling in some cells. Examination of 12 subclones derived from two cloned transfectants, however, revealed a poor correlation between spontaneous metastatic potential and intercellular junctional coupling. The results suggest that loss of junctional communication between cells is often but not always associated with the progression of cells from benign to metastatic states.

Development and characterization of a syngeneic monoclonal antibody to a rat mammary tumor metastasis-associated mucin-like cell-surface antigen (gp580).

A rat hybridoma producing IgM monoclonal antibody (MAb) GP21:56 was generated with specificity for a high-molecular-weight, mucin-like glycoprotein (gp580) present on highly metastatic 13762NF rat mammary adenocarcinoma cells. The hybridoma was made by fusing rat Y3 Ag1.2.3 myeloma cells with spleen cells from a rat immunized i.d. with purified gp580. The gp580 appeared to be of low immunogenicity in syngeneic F344 rats because a total of 27 fusions were required to produce one hybridoma with specificity for this glycoprotein. Immunoblotting of purified gp580 after electrophoresis in 1% agarose and antibody-binding assays using purified gp580 linked to microtiter plates confirmed that MAb GP21:56 bound specifically to gp580. Other MAbs made against breast mucins were negative for gp580 reactivity. Enzyme-linked immunoabsorbent assays (ELISA) and radiolabelled antibody binding assays demonstrated that MAb GP21:56 bound to 13762NF adenocarcinoma cell lines and clones in relation to their spontaneous metastatic potentials; significantly more MAb GP21:56 bound to highly metastatic MTLn3 cells than to low metastatic MTC cells, and MAb GP21:56 showed little reactivity towards the majority of other cell lines tested, whether of rodent or of human origin. Kinetic binding studies indicated that MAb GP21:56 does not have a high affinity for gp580 but, once bound, it shows high avidity for this sialogalactoprotein. Localization studies using frozen tissue sections of 13762NF tumors indicated that MAb GP21:56 reacts with tumor cells grown in vivo in an analogous manner to in vitro cultured cells. Using immunoperoxidase techniques, less than 50% of the highly metastatic MTLn3 tumor cells were stained, whereas approximately 20% of the intermediate metastatic MTF7 and MTLn2 cells and less than 10% of low metastatic MTC and MTPa cells were stained with MAb GP21:56. The cell-to-cell reactivity was heterogeneous and mainly associated with the tumor-cell surface and extracellular matrix.

Identification and partial characterization of a nucleolar antigen with a molecular weight of 145,000 found in a broad range of human cancers.

Previous studies in our laboratory have indicated the presence of nucleolar antigens in tumors which were not detected in normal tissues. Some of the polyclonal antisera produced in these studies were shown to identify a Mr, 145,000 nucleolar antigen on immunoblots of tumor nucleoli but not in normal human liver nucleoli. A monoclonal antibody to a Mr 145,000 nucleolar protein (p145) was produced by immunization of mice with a nucleolar extract of HeLa cells which is enriched with this antigen. The monoclonal antibody showed bright nucleolar immunofluorescence localization in a broad range of human tumors including cancers of the gastrointestinal tract, genitourinary tract, lung, liver, muscle, cartilage, and blood. The p145 nucleolar antigen was not detected in most normal human tissues or in benign tumors, with only weak nucleolar staining observed in spermatogonia of the testes and in ductal regions of some hypertrophied prostates. Nucleolar antigen p145 was extracted from HeLa cell nucleoli by homogenization in a 0.01 M Tris buffer containing 0.2% deoxycholate. On sucrose density gradient centrifugation, the antigen remained sedimented with the nucleolar ribonucleoprotein fraction. Nucleolar antigen p145 was released from ribonucleoproteins following treatment with 4 M guanidinium hydrochloride or RNase. Peptide mapping of nucleolar antigen p145 showed that it was distinct from other known nucleolar antigens. Although it remains to be determined if the p145 antigen plays a role in cell transformation, maintenance of the malignant phenotype, or in cell division, it may have value as a tumor marker or as a therapeutic target.

Electrophoretic analysis of HeLa cell and human liver nucleolar proteins and antigens.

The major antigens in HeLa nucleolar extracts recognized by immunoblots with rabbit antisera had molecular weights of 145, 110, and 34 kd. The major antigens in the HeLa nucleolar residues had molecular weights of 145, 110, 86, 68, 55, 48, and 34 kd. In the liver, the major antigens in the nucleolar extracts had molecular weights of 86 and 76 kd. In the liver residues, the major antigens had molecular weights of 110, 86, 76, 65, and 55 kd. On two-dimensional gels stained with Coomassie blue, the HeLa nucleolar extract contained large amounts of protein B23 and lesser amounts of protein C23. In the liver nucleolar samples separated on two-dimensional (2-D) gels, protein 55/7.6 (Mr/pI) was the major protein in the extract. Lesser amounts of protein B23 were identified. Addition of protease inhibitors markedly improved the quality of the protein samples as shown in one-dimensional gel patterns for liver nucleolar proteins and to a lesser extent for HeLa nucleolar proteins. In the 2-D immunoblots of the HeLa extract and HeLa residues, the major stained band was protein C23 (110/5.2-5.6). In the liver extract, the major bands were 70/5.8-7 and 60/5.8-7. With a monoclonal antibody (MS-3) to protein C23 a 76/5.8 band was more notable in the residue which supported the results of the Coomassie blue and immunostains with rabbit antinucleolar antibodies. Some degradation products of protein C23 were observed in both the liver extract and the liver residue despite the use of protease inhibitors. Protein C23 along with other proteins are major antigens in HeLa nucleoli. In human liver nucleoli, a major protein 55/7.6 was identified which was not observed in the HeLa extracts. These studies show that a combination of protease inhibitors markedly reduced degradation of proteins in liver samples and provided a more satisfactory sample for comparison with HeLa cells. Qualitative and quantitative differences were found in the nucleolar proteins by Coomassie blue and immunostaining.

Fibrillarin: a new protein of the nucleolus identified by autoimmune sera.

Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.

Purification and partial characterization of ring-shaped miniparticles.

Small ring-shaped miniparticles, found in the nucleus nucleolus and cytoplasm of a variety of normal and malignant cells, have been purified by a procedure involving differential and gradient centrifugation, hydroxylapatite chromatography and preparative electrophoresis. They are multisubunit proteins comprised of at least six polypeptide chains of Mr 20,000-30,000 Daltons (by one-D SDS-PAGE which present a series of 20-30 spots on two-D IEF/SDS gels. They can be reversibly dissociated in EDTA containing buffers. They are not membrane associated and contain no carbohydrate. They have an S20,W of 21S on sucrose gradients. They are immunologically similar in several human cell lines.

Comparison of nuclear proteins of several human tumors and normal cells by two-dimensional gel electrophoresis.

Using isoelectric focusing and sodium dodecyl sulfate two-dimensional gel electrophoresis, 9 M urea-extractable nuclear proteins from four human tumor cells (HeLa, Namalwa, acute myelogenous leukemia, and lymphoma) and four normal human cells (IMR-90, WI-38, liver, and lymphocytes) were compared. Two protein spots, 140/7.7 and 54/6.6, were found in all four tumor cells but not in the four normal cells studied. Two protein spots, 56/6.7 and 56/6.9, were found in all four normal cells but not in any of the tumors studied. None of these proteins was common to those found in the earlier studies on rat tumors (H. Takami et al., Cancer Res., 39: 2096-2105, 1979).

Two-dimensional gel electrophoresis of nuclear phosphoproteins of Novikoff hepatoma and regenerating liver.

Two-dimensional polyacrylamide gel electrophoresis patterns of 32P-labeled nuclear proteins in Novikoff hepatoma and regenerating liver relatively small number of the many nuclear protein spots were phosphorylated. The major phosphoprotein spots differed in size and shape from the stained protein spots. Seven phosphoproteins, 125/5.8, 125/7.2, 100/5.9, 85/5.9, 56/5.1, and 50/5.1 (mol. wt. x 10-(3)/pI in the Novikoff hepatoma nuclei were not found in the 18-h regenerating liver nuclei. One nuclear phosphoprotein, 54/6.5, was found in the liver but not in the hepatoma. The four major phosphoproteins, 45/5.3-6.0, 40/5.3-6.0, 35/5.3-6.0, and 25/5.8-6.8 containing about 60% of the total 32Pi of the nuclear proteins, were found in both types of cells. These proteins were of unusual density and shape. Some proteins, such as 125/5.8 and 85/5.9, were enriched in 0.01 M tris extract of the tumor, and proteins 125/7.2 and 56/6.1 were enriched in 0.35 M NaCl extract of the tumor.

Enrichment of special Novikoff hepatoma and regenerating liver mRNA by hybridization to cDNA-cellulose.

Total polysomal poly(A)+ RNA of Novikoff hepatoma and of 18-hr regenerating rat liver were compared by analysis of their in vitro translational products on two-dimensional isofocusing/sodium dodecyl sulfate gels. This technique resolved the translated proteins sufficiently to permit detection of quantitative and some qualitative differences between the two mRNA populations. Excess cDNA from regenerating liver or Novikoff hepatoma, covalently linked to cellulose, was used to adsorb the complementary mRNA sequences from Novikoff hepatoma or regenerating liver. As shown by two-dimensional gel electrophoresis, the translated products of the bound mRNA fractions contained proteins common to both tissues. Novikoff hepatoma mRNA which did not bind to regenerating liver cDNA was enriched in sequences encoding for proteins 11/5.1, 15/6.8, 40/8.2, and 65/5.1 (shown as molecular weight/pI). These polypeptides were not detectable in the translational products of regenerating liver mRNA. Regenerating liver mRNA that was not bound to Novikoff hepatoma cDNA was enriched in sequences coding for proteins 12.5/4.9, 13.5/7.4, 17/8.2, 24/5.5, and 46/6.4; these proteins were not found in the translational pattern from Novikoff hepatoma. These results show that adsorption of mRNA to solid-phase cDNA provides a valuable technique for differentiating mRNA species in related tissues and for corresponding enrichment of these specific mRNAs.

Adsorption of messenger RNA of Novikoff hepatoma, normal liver, and regenerating liver on complementary DNA-cellulose affinity matrices.

Complementary DNA (cDNA)-oligodeoxythmidylate-celluloses were prepared from cDNA copies of polysomal messenger RNA (mRNA) of Novikoff hepatoma, normal rat liver, and regenerating rat liver. cDNA synthesis with reverse transcriptase was approximately 46% with respect to input mRNA with oligodeoxythymidylate-cellulose primer. The cDNA's of normal liver, regenerating liver, and Novikoff hepatomas were used as affinity matrices for hybridization of different mRNA species. Under the conditions used, degradation of mRNA was not detected. After normalization for homologous hybridization efficiency, 53 and 65% of the Novikoff hepatoma mRNA bound to normal liver and regenerating liver cDNA's. Under these conditions an average of 82% of mRNA of normal liver bound to regenerating liver cDNA, and 92% of regenerating liver mRNA bound to normal liver cDNA. The bound and unbound mRNA's were analyzed by translation in the wheat germ system; 2-D gel analysis of the proteins synthesized in the wheat germ system indicated that the cDNA affinity columns selectively adsorbed some mRNA species.

Comparative studies on the '5'-cap' and in vitro translational activity of cytoplasmic and nuclear poly A(+) RNA1.

The translational activities of cytoplasmic poly A(+)RNA of normal rat liver and Novikoff hepatoma cells in the wheat germ cell free system were found to be approximately 15-20 times greater than tose of the corresponding nuclear poly A(+) RNA. The translationsl activities were 85 and 62 pmoles 3H-leucine incorporated/micron g cytoglasmic poly A(+) RNA for the liver and tumor respectively and 3-4 pmoles 3H-leucine incoporated/micron g nuclear poly A(+)RNA. Inasmuch as intergity of the '5'-cap' of mRNA is essential for its translational activity, quantitative comparisons were made of its content in these RNA fractions. Of the total 32P incorporated into the tumor cytoplasmic poly A(+) RNA, 0.41% was in the '5'-cap'; in nuclear poly A(+) RNA, the '5'-cap' contained 0.11%. After periodate oxidation and labeling with KB3H4, m7 guanosine, the 5'-terminal nucleoside in both liver and Novikoff hepatoma nuclear poly A(+) RNA contained approximately 20% as much isotope as in the cytoplasmic poly A(+) RNA. These results suggest the lower translational activity of nuclear poly A(+) RNA is partly related to its lower content of the '5'-cap'. Molecular selection of poly A(+) RNA for transport out of the nucleus or further cytoplasmic processing may account for the higher percentage of the '5-cap' and the greater translational activity of the cytoplasmic poly A(+) RNA. During these studies, it was also found that the m7 guanosine of the '5'-cap' was not removed during translation of the mRNA in the wheat germ system; this result suggests that the '5'-cap' may associate with allosteric binding sites of initiation factor(s).

Detergent lysis for isolation of intact polysomes of Nivikoff hepatoma ascites cells.

An efficient, rapid procedure for the isolation of polysomes from Novikoff hepatoma ascites cells that avoids mechanical shear was developed, and Ivory was used as the detergent to lyse the cells. This method yields classes of large polysomes and 3- to 5-fold increased yields over that of routine homogenization procedures. Up to 70% of the polysome population is larger than tetramers. The polysomes and ribosomes are morphologically intact and contain undegraded ribosomal RNA and biologically active messenger RNA. This procedure also yields nuclei free of cytoplasmic contamination as a by-product of polysome isolation. These nuclei do not show evidence of protein degradation and are enzymatically active with respect to RNA polymerases I and II.

Characterization of nuclear and chromatin poly A-containing RNA.

Poly A-containing RNA was isolated from Novikoff hepatoma cell chromatin on nitrocellulose membrane filters after the cells were labeled with 32P-orthophosphate for 6 hours. Under denaturing conditions, the poly A-containing chromatin RNA had a sedimentation coefficient of approximately 8-18S. The presence of poly A tracts in the chromatin RNA fractions indicate that poly A addition occurs almost immediately after transcription. Poly A-containing chromatin RNA and nuclear RNA were approximately 1/5 as active as cytoplasmic poly A-containing RNA in the wheat germ translational system. Since chromatin poly A-containing RNA is the precursor of cytoplasmic poly A-containing RNA, further processing of this RNA prior to its attachment to polysomes may account for its increase in translational capacity.