Thyroid targeting of the N-ras(Gln61Lys) oncogene in transgenic mice results in follicular tumors that progress to poorly differentiated carcinomas.

Ras oncogenes are frequently mutated in thyroid carcinomas. To verify the role played by N-ras in thyroid carcinogenesis, we generated transgenic mice in which a human N-ras(Gln61Lys) oncogene (Tg-N-ras) was expressed in the thyroid follicular cells. Tg-N-ras mice developed thyroid follicular neoplasms; 11% developed follicular adenomas and approximately 40% developed invasive follicular carcinomas, in some cases with a mixed papillary/follicular morphology. About 25% of the Tg-N-ras carcinomas displayed large, poorly differentiated areas, featuring vascular invasion and forming lung, bone or liver distant metastases. N-ras(Gln61Lys) expression in cultured PC Cl 3 thyrocytes induced thyroid-stimulating hormone-independent proliferation and genomic instability with micronuclei formation and centrosome amplification. These findings support the notion that mutated ras oncogenes could be able to drive the formation of thyroid tumors that can progress to poorly differentiated, metastatic carcinomas.

Identification of a new subset of cells exhibiting dendritic phenotypes in patients affected by breast proliferative disorders.

We report that a subset of circulating cells reacting with a monoclonal antibody raised against a protein marker is significantly increased in the peripheral blood of women carrying benign or malignant breast diseases, particularly in patients under 55 years of age with ductal mammary carcinomas. These cells were statistically (confidence level of 99%) less represented in a control population including healthy women or women carrying carcinomas of origin other than breast. Double staining analysis showed that they harbor markers of dendritic cells and exhibit endo- cytic activity, as determined by their ability to internalize FITC-dextran particles. Their dendritic morphology was further demonstrated by electron microscopy of sorted antibody-positive cells. However, expression of surface molecules, such as CD34 and CD14, usually not present in differentiated populations of dendritic cells was also observed. Adherent cells of patients with breast ductal carcinoma including mostly cells of this new subset were efficient stimulators of mixed lymphocyte reaction, attaining maximal stimulatory activity attained after TNFalpha treatment. In conclusion, we have shown that a subset of cells characterized by a phenotype suggestive of a yet undescribed stage of maturation of the dendritic cell lineage is accumulated in the blood of patients affected by breast proliferative disorders.

Design of affinity tags for one-step protein purification from immobilized zinc columns.

Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

Diamine oxidase (DAO) activity increase in sera of tumor patients: an unreliable marker.

Circulating diamine oxidase (DAO) (EC 1.4.3.6) activity was determined in the sera of patients with different malignant tumors. The increase in this activity only partially reflected tumor activity so it is presumably unreliable as a diagnostic marker. The increase in activity may be linked to the tumor location: the lung and intestine are rich in DAO so the rise cannot be considered a characteristic consequence of the tumor's activity. Besides, increases in DAO activity have also been observed in non-neoplastic diseases.

Polyamines and nucleic acids in gestational trophoblastic tumors.

To test the relationship between tumor malignancy and content and distribution of polyamines and nucleic acids, 2 forms of human gestational trophoblastic tumors were examined: the hydatidiform mole (self-limited form) and the human chorio-carcinoma (invasive form) xenografted into nude mice. The results indicate that there are 2 significant differences between the choriocarcinoma and the mole: 1) the choriocarcinoma is characterized by increased polyamine and nucleic acid levels, 2) tissues differ in their putrescine:spermidine and spermidine:spermine ratios. There is an increase in polyamines in the urine of mole-bearing patients over that of normal controls. The correlation between putrescine and spermidine with the chorionic gonadotropin indicates that these 2 polyamines reflect the biological activity of the mole.

Vector engineering anomalies: impact on fusion protein purification performance.

Recombinant protein purification using IMAC is often carried out by protein fusion to affinity tags. We have identified several tags useful for protein purification on Zn(II)-IDA columns. These tags were fused to the green fluorescent protein (rGFPuv) using the vector pGFPuv distributed by Clontech Lab (Palo Alto, CA) and analyzed for purification on Zn(II)-IDA. Each fusion protein exhibited elution heterogeneity (elution in two distinct pHs) from Zn(II)-IDA columns This led us to believe that two populations of fluorescent proteins were being expressed: one without the tag coeluting with Escherichia coli proteins at pH 7.5 and one bearing the tag eluting at a pH lower than pH 7.5. Assessment of the constructs revealed the possibility of a ribosomal binding site and start codon between the fusion tag and the rGFPuv sequence which might be used as a secondary translation start site. This hypothesis was confirmed by changing the second ATG (methionine) codon to an ACG (threonine) codon. The protein produced from this new construct eluted in a single fraction from a Zn(II)-IDA column. Thus, vector irregularities (along with other possibilities) should be examined when searching for the cause of elution heterogeneity of a target protein.

Coordination of two high-affinity hexamer peptides to copper(II) and palladium(II) models of the peptide-metal chelation site on IMAC resins.

The coordination of peptides Ser-Pro-His-His-Gly-Gly (SPHHGG) and (His)6 (HHHHHH) to [PdII(mida)(D2O)] (mida2- = N-methyliminodiacetate) was studied by 1H NMR as model reactions for CuII(iminodiacetate)-immobilized metal affinity chromatography (IMAC) sites. This is the first direct physical description of peptide coordination for IMAC. A three-site coordination is observed which involves the first, third, and fourth residues along the peptide chain. The presence of proline in position 2 of SPHHGG achieves the best molecular mechanics and bonding angles in the coordinated peptide and enhances the interaction of the serine amino nitrogen. Histidine coordination of H1, H3, and H4 of (His)6 and H3 and H4 of SPHHGG was detected by 1H NMR contact shifts and H/D exchange of histidyl protons. The EPR spectra of SPHHGG and HHHHHH attached to the [CuII(mida)] unit were obtained for additional modeling of IMAC sites. EPR parameters of the parent [Cu(mida)(H2O)2] complex are representative: gzz = 2.31; gyy = 2.086; gxx = 2.053; A parallel = 161G; AN = 19G (three line, one N coupling). Increased rhombic distortion is detected relative to the starting aqua complex in the order of [Cu(mida)L] for distortion of HHHHHH > SPHHGG > (H2O)2. The lowering of symmetry is also seen in the decrease in the N-shf coupling, presumably to the imino nitrogen of mida2- in the order 19 G (H2O), 16 G (SPHHGG) and 11 G (HHHHHH). Visible spectra of the [Cu(mida)(SPHHGG)] and [Cu(mida)(HHHHHH)] as a function of pH indicate coordination of one histidyl donor at ca. 4.5, two in the range of pH 5-7, and two chelate ring attachments involving the terminal amino donor for SPHHGG or another histidyl donor of HHHHHH in the pH domain of 7-8 in agreement with the [PdII(mida)L] derivatives which form the two-chelate-ring attachment even at lower pH as shown by the 1H NMR methods.

Reversion of a transcriptionally defective MHC class II-negative human B-cell mutant.

RJ2.2.5, a mutant derived from the human B-lymphoma cell, Raji, is unable to express the MHC class II genes because of a recessive transcriptional defect attributed to the lack of an activator function. We report the isolation of a RJ2.2.5 revertant, namely AR, in which the expression of the mRNAs encoded by these genes is restored. Comparison of the binding of nuclear extracts or of partially purified nuclear preparations from the wild-type, the mutant and the revertant cells to a conserved MHC class II promoter element, the X-box, showed no alteration in the mobility of the complexes thus formed. However, in extracts from RJ2.2.5, and other MHC class II negative cell lines, such as HeLa, the amount of complex observed was significantly higher than in wild-type Raji cells. Furthermore, the binding activity exhibited by the AR revertant was lower than that of the RJ2.2.5 and higher than that of Raji. The use of specific monoclonal antibodies indicated that in all cases c-Jun and c-Fos or antigenically related proteins were required for binding. An inverse correlation between the level of DNA-protein complex formed and the level of MHC class II gene mRNA expressed in the three cell lines was apparent, suggesting that overexpression of a DNA binding factor forming complexes with class II promoter elements may cause repression of MHC class II transcription. A model which reconciles the previously ascertained recessivity of the phenotype of the mutation carried by RJ2.2.5 with the findings reported here is discussed.

Circulating antibodies against the breast tumor marker GCDFP-15/gp17 in mammary carcinoma patients and in patients carrying benign breast conditions.

Sera samples from 111 women, including 73 breast cancer patients and 38 patients with benign diseases of the breast, were examined. These were compared with samples from healthy women or from patients carrying tumors of origin other than breast as controls. This was done to determine whether antibodies against GCDFP-15/gp17, a protein of gross cystic disease fluid also secreted by mammary apocrine tumor cells, could be found. We observed that 2.6% of mammary disease patients affected by benign conditions and 5.5% of patients carrying malignant mammary gland tumors expressed statistically significant amounts of antibodies against GCDFP-15/gp17 (p < 0.01). The highest circulating anti-GCDFP-15/gp17 antibody levels occurred in patients with highly malignant ductal or lobular carcinoma of the breast and in patients with dysplasia. No correlation was found between the presence of circulating antibodies and the size of the tumor or the age of the patients. A bimodal correlation with the percent of invaded lymph nodes was observed instead. IgM and IgG isotypes were detected among the circulating anti-GCDFP-15/gp17 antibodies, suggesting the involvement of a T-cell-mediated immunoresponse. Our findings raise the possibility that the anti-GCDFP-15/gp17 immune response may be useful as a tool for investigating some aspects of the mechanisms of breast disease progression and that GCDFP-15/gp17 may be explored as an antigen for anti-tumor vaccination. Int. J. Cancer (Pred. Oncol.) 84:568-572, 1999.