Molecular markers in Paget disease of the breast.

BACKGROUND AND OBJECTIVES: Molecular markers are increasingly being analyzed in tumor specimens because of their relevance to both prognosis and choice of therapy. Paget disease of the breast is an uncommon form of breast cancer, in which molecular markers have not been well characterized. The objective of this study was to investigate the expression of c-erbB-2, p53, Ki-67, Cyclin D1, Bcl-2, estrogen receptors (ER), and progesterone receptors (PR) in mammary Paget disease. METHODS: Archival tumor tissues from 14 patients diagnosed between 1990 and 1999 with Paget disease of the breast were analyzed for these molecular markers by using an automated immunohistochemical assay. Both the intraepidermal Paget cells and the underlying carcinoma were assessed for these markers. RESULTS: The majority of Paget cells were positive for c-erbB-2 (92.9%), Cyclin D1 (100%), and Ki-67 (85.7%), but very few were positive for Bcl-2 (14.3%). p53 was overexpressed in 42.9% of the cases, and only 28.6% were positive for ER and PR. The rate of expression of these biologic markers was similar in both the Paget cells and the underlying intraductal and/or ductal carcinoma cells. CONCLUSIONS: Tumors from patients with Paget disease of the breast were positive for c-erbB-2, Cyclin D1, and Ki-67, molecular markers commonly associated with more aggressive tumor behavior and poorer survival in breast cancer patients. Few of these tumors expressed Bcl-2 or ER and PR, which are generally associated with a better prognosis. Similar expression of these markers in both Paget cells and the underlying carcinoma supports the theory that these cells are the result of an intraepidermal spread of ductal carcinoma.

Interferon alpha2b inhibits the murine melanoma cell line Cloudman S91 in vivo but not in vitro: a model for studying tumor cell-cytokine interactions.

Interferon alpha2b has recently been shown to improve outcome in patients with metastatic malignant melanoma. The high-dose interferon therapy used is however associated with significant systemic adverse effects. These adverse effects are likely related to the multitude of actions of interferon which in addition to its antineoplastic effects also possesses antiviral and immunomodulating properties. Elucidation of the mechanism of the antiproliferative effects of interferon may allow for the development of agents that possess the antineoplastic properties while being devoid of the other effects that make interferon toxic. In the animal model developed for this study tumors in mice receiving interferon alpha2b grew at a slower rate and achieved a small final tumor volume (3040 +/- 690 vs 1400 +/- 314 mm3 for the control and treated groups respectively, P < 0.05). Furthermore the final tumor weight in the treated group was significantly smaller (1.50 +/- 0.21 g vs 2.76 +/- 0.46 g for the treated and control groups respectively; P = 0.036). The (3-[4,5-Dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide) (MTT) colorimetric assay failed to reveal any direct effects of interferon alpha2b on this murine melanoma cell line. This antiproliferative effect of interferon alpha2b was in addition found to be independent of alterations in the expression of the angiogenic cytokines vascular endothelial growth factor, basic fibroblast growth factor, and transforming growth factor beta.

Bromodeoxyuridine improves the cytotoxic effect of cisplatin: a comparison with 5-fluorouracil.

We compared the effects of the radiosensitizers, 5-bromo-2'-deoxyuridine (BUdR) and 5-fluorouracil (5-FU) alone and in combination and cis-diamminedichloroplatinum (cisplatin, DDP) on the growth of B16 amelanotic melanoma (B16a) tumors in mice. In a preliminary study, tumor growth was significantly inhibited in the presence of BUdR and was further reduced with the combination of BudR and DDP. In a second experiment, BUdR was found to be more effective than 5-FU when used in combination with DDP. At the completion of the study, tumor volumes as a percentage of control values in mice treated with a single drug were as follows: 5-FU (50 mg/kg per day for 7 days) 76.5% (P < 0.05), BUdR (100 mg/kg per day for 7 days) 68% (P < 0.05) and DDP (5 mg/kg x 3) 54% (P < 0.01). Combining 5-FU and DDP at these dosages reduced volumes to 38% (P < 0.01), while BUdR + DDP-treated mice had tumor volumes only 28% (P < 0.001) the size of untreated controls. Furthermore, the toxicity, as demonstrated by a decrease in body weight and an increase in mortality, was more severe in mice receiving 5-FU than in those receiving in BUdR. DDP interacts synergistically with either BUdR or 5-FU in its cytotoxic action in vivo. No such relationship could be demonstrated in vitro, suggesting that the pharmacologic activity of these drugs may be responsible for the antitumor activity than direct cytotoxic effects. We propose that BUdR is more effective than 5-FU as a potentiator of DDP in this murine melanoma model.

Identification of the alpha IIb beta 3 integrin in murine tumor cells.

Previously, we have identified an alpha IIb beta 3-like integrin in tumor cells by using antibodies against platelet alpha IIb beta 3. However, alpha IIb beta 3 was considered to be expressed strictly in megakaryocyte lineage cells. In order to resolve this controversy, the alpha IIb beta 3-like integrin in murine B16 amelanotic melanoma (B16a) cells was characterized at DNA, RNA, and protein levels. The presence of alpha 5, alpha v, alpha IIb, beta 1, and beta 3 genes in B16a cells was confirmed by Southern analysis. mRNAs for all these integrins except alpha v were detectable by Northern blotting. The alpha IIb beta 3 protein was identified by Western blotting using subunit-specific antibodies and by immunoprecipitation using complex-specific antibody. The alpha IIb beta 3 integrin was localized intracellularly by immunocytochemistry. Finally, alpha IIb and beta 3 mRNAs were amplified by reverse transcription-polymerase chain reaction and the identity of alpha IIb was verified by sequencing. Partial DNA and deduced amino acid sequence analysis showed that B16a alpha IIb shares approximately 80% homology with the human alpha IIb and approximately 90% homology with the rat alpha IIb, whereas B16a alpha IIb shares only approximately 26% homology with the human alpha v. These experiments indicate that the alpha IIb beta 3-like protein in B16a cells is the authentic alpha IIb beta 3 and demonstrate, for the first time, that integrin alpha IIb beta 3 is not confined to megakaryocyte lineage cells.

HLA-DR is a procoagulant.

Numerous incidents of thromboembolic complications have been documented in cancer patients and in recipients of mismatched organ transplants. Tumor procoagulants have also been implicated in the process of metastasis. Two protein bands of 35,000 and 28,000 daltons isolated from human ovarian carcinoma possessed procoagulant activity. The 35,000 dalton protein had an amino terminal sequence identical to that of the major histocompatibility antigen HLA-DR. Further, isolation of the protein using immunoaffinity column chromatography with monoclonal antibody to HLA-DR resulted in the isolation of procoagulant activity. The immunoaffinity purified protein enhanced thrombin generation in recalcified normal plasma approximately 20- fold. HLA-DR procoagulant activity was completely inhibited by Staphylococcal enterotoxin A. We propose that the procoagulant nature of HLA-DR may contribute to thrombotic disorders in several cancers and in association with graft rejection. The ability of enterotoxin A to inhibit this procoagulant may lead to development of future therapeutic strategies.

Thrombin-reactive polypeptides of platelets may regulate inhibition of thrombin by antithrombin.

The central enzyme involved in blood coagulation and activation of platelets is the serine proteinase thrombin. The principal inhibitor of this proteinase in plasma is antithrombin. The mechanism of regulation of the thrombin-antithrombin reaction remains unknown. Two polypeptides of 74 and 55 kDa present on the platelet surface and in plasma are known to specifically enhance the activity of thrombin on different substrates. This study was undertaken to assess the effects of these platelet proteins on thrombin-antithrombin interaction. Direct measurements of residual thrombin activity in mixtures of thrombin and antithrombin, in the presence or absence of the platelet proteins, were made utilizing a specific chromogenic substrate. Under these conditions, when 60% of thrombin activity was inhibited by antithrombin in controls, 100% of enzyme activity was retained in the presence of the platelet proteins. When heparin was used in these assays, the rate of inhibition of thrombin by antithrombin was much more rapid and 62% of thrombin activity remained after 1 min. Under these conditions, the platelet proteins continued to protect thrombin from inactivation with 98% activity remaining at 1 min and 85% activity at 5 min. In contrast, the inhibition of trypsin by antithrombin was not affected by the platelet proteins. Additional studies in platelet aggregation showed that the platelet polypeptides have two effects on thrombin: (i) protection of the enzyme inhibition by antithrombin and (ii) stabilization of thrombin from loss of activity due to aging. The results suggest a novel role for the platelet proteins in hemostasis - regulation of the inhibition of thrombin by antithrombin.

Protein phosphorylation and activation of platelets by wheat germ agglutinin.

The incorporation of 32Pi into the 47 and 20 kDa polypeptides in platelets activated by wheat germ agglutinin (WGA) was studied. The pattern of enhanced phosphorylation produced by the lectin was comparable to that by thrombin. The 47 kDa polypeptide was phosphorylated at both serine and threonine while the 20 kDa protein was mainly labeled at serine residues. However, the ratio of phosphoserine to phosphothreonine in the 47 kDa polypeptide in WGA-activated platelets was higher than thrombin-stimulated platelets. Addition of N-acetylglucosamine at different times blocked platelet activation by WGA. There was a concomitant modification in the phosphorylation of the 47 kDa protein. These data suggest that the phosphorylation of the 47 kDa polypeptide may modulate the WGA-receptor mediated activation of platelets. Our studies also demonstrate that activation of platelets by different stimuli may lead to differential phosphorylation of different amino acid residues in the same protein.

Thrombin-reactive polypeptides of human blood. Some biochemical and immunological properties.

Two polypeptides of 74 kDa and 55 kDa have been isolated from human platelets by immunoaffinity and lectin affinity chromatography and their effects on thrombin reactivity have been examined. These proteins in combination enhanced the aggregation of platelets by thrombin while aggregation induced by trypsin, collagen and adenosine diphosphate was not significantly affected. An enhancement in the action of thrombin on fibrinogen, N-benzoylarginine ethyl ester and H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride was also observed in the presence of the platelet proteins. Under similar conditions, the proteins did not influence the esterolytic activity of trypsin or plasmin. Studies at different thrombin and protein concentrations showed maximum enhancement of enzyme reactivity when the ratio between the peptides and thrombin was optimal. In the presence of these proteins, the affinity of thrombin for N-benzoylarginine ethyl ester was about twofold higher than in the control. Two polypeptides with properties similar to those described above have also been isolated from human plasma. Antibodies to the above proteins isolated from either platelets or plasma were raised in rabbits. Intact platelets solubilized in Triton X-100 or plasma showed two precipitin lines in immunoelectrophoresis against both of the above antisera and a similar pattern was observed with the isolated polypeptides. The polypeptides did not interact in immunoelectrophoresis with antisera to whole serum, antithrombin, C4 binding protein or protein S. These 74-kDa and 55-kDa polypeptides contained radioactivity when radioiodinated platelets were used suggesting that they are located on the cell surface. Fresh plasma was analyzed by gel electrophoresis under nondenaturing and denaturing conditions and the proteins were transferred to nitrocellulose sheets. Staining with antibody to these thrombin-reactive proteins and 125I-protein A showed several reactive plasma proteins under nondenaturing conditions with the major band migrating in the albumin area. In plasma treated with sodium dodecyl sulfate, the 74-kDa and 55-kDa components were observed. A prominent 74-kDa band and a fainter 55-kDa component were again observed when platelets solubilized in sodium dodecyl sulfate were analysed by the above procedure. It is proposed that human platelets and plasma contain polypeptides which may directly modulate thrombin reactivity.

A novel thrombin enhancement factor in human plasma.

A protein has been isolated from human plasma by gel filtration followed by affinity chromatography with a derivative of wheat germ agglutinin and ion exchange chromatography. This protein showed one peak in high performance liquid chromatography but in gel electrophoresis, in the presence of sodium dodecyl sulfate and beta-mercaptoethanol, revealed two major components of 74 kDa and 55 kDa. These results indicate that the protein probably exists as a complex of the two polypeptides. This protein complex enhanced platelet aggregation by thrombin while aggregation induced by ADP was not significantly affected. Similarly, the rate of thrombin action on fibrinogen and N-benzoylarginine ethyl ester as measured in a spectrophotometer was increased in the presence of this plasma protein. These results suggest the presence of a protein complex in human plasma which can directly interact with thrombin and enhance its reactivity.

In vivo studies of serum C-reactive protein turnover in rabbits.

We determined the plasma half-life of the acute phase protein C-reactive protein (CRP) both in normal rabbits and in rabbits that had received inflammatory stimuli. Rabbit CRP was purified from acute phase serum by Cx-polysaccharide affinity chromatography, radiolabeled, and rendered pyrogen-free. Six unstimulated rabbits were injected intravenously with (125)1-CRP prepared by the lactoperoxidase method and four were injected with CRP labeled by methylation using [(14)C]formaldehyde. Blood samples were obtained at 0.25 h and at intervals thereafter. Plasma half-life of CRP was calculated from the data generated during the first 12 h, by which time an average of 86% of labeled protein had disappeared from the blood stream. The mean half-life for CRP was 4.45+/-0.2 h, with no significant difference (0.40 < P < 0.45) between (125)1- and (14)C-labeled CRP. In six animals stimulated with either endotoxin or turpentine 24 h before injection of labeled CRP, a mean half-life of 5.8+/-0.6 h was found, not significantly different (0.30 < P < 0.35) from unstimulated rabbits. We equated fractional catabolic rate to fractional disappearance rate, since the rate constant for passage of CRP from vascular to extravascular compartment can be assumed to be relatively small compared to the observed fractional disappearance rate. Fractional catabolic rate was independent of serum CRP concentration; average fractional catabolic rate in all 16 animals was 14+/-0.8% h(-1) of the plasma pool. We were able to estimate rate of CRP synthesis, based on steady-state assumptions of pool sizes in those rabbits whose serum CRP levels did not change substantially during the period of study. Values as low as 6.7 mug/kg per h in the unstimulated animals and as high as 560 mug/kg per h in the stimulated animals were found.

A novel thrombin-reactive protein complex in human platelets.

Attempts were made to isolate a 74,000-dalton protein which specifically and competitively blocked platelet aggregation by thrombin (Ganguly, P., and Fossett, N. G. (1981) Blood 57, 343-352) by two independent affinity chromatography methods. The protein isolates showed a main band migrating slightly faster than albumin in gel electrophoresis under nondenaturing conditions. In the presence of sodium dodecyl sulfate, electrophoresis of protein preparations consistently revealed the presence of four polypeptides of 74,000, 55,000, 27,000, and 20,000 daltons. The results suggest that these polypeptides are probably present as a multiprotein complex in platelets. The 55,000-dalton protein, like the 74,000-dalton protein, inhibited thrombin-induced platelet aggregation, while aggregation induced by adenosine diphosphate or trypsin was not significantly affected. However, incubation of the 55,000-dalton protein with the 74,000-dalton protein prior to the addition of thrombin did not inhibit platelet aggregation by thrombin, while the individual components under the same conditions did. In fact, the two proteins in combination could significantly enhance platelet aggregation by thrombin. These results suggest the existence of a multiprotein complex in human platelets, the components of which may modulate the action of thrombin on platelet aggregation.

N-terminal amino acid sequence of proalbumin from inbred buffalo rats.

The sequence of radioactively labelled amino acids at the N-terminus of proalbumin was determined by automated Edman-degradation. [3H] Valine, [3H]phenylalanine or [14C]arginine was incorporated into protein in vivo for a time period of 10 min after injection. Since albumin remains unlabelled during this time period (Urban et al., 1976), separation of proalbumin and albumin was not required for this work. Hence, compared to previous methods, a shorter purification procedure could be used which increased the yield of anti-albumin-precipitable protein and reduced the risk of proteolysis. Microsomes were prepared from livers removed 10 min after injection of the radioactively labelled amino acids. A buffer extract of the acetone-dried powder from these microsomes was chromatographed on DEAE-cellulose. All protein obtained after chromatography which could be precipitated with antiserum to serum albumin was isolated by immunoprecipitation and subsequent separation of the antigen-antibody complex. The sequence of radioactive amino acids in this antigen preparation suggests that about 20-25% of proalbumin possessed at the N-terminus the pentapeptide sequence X-Val-Phe-Arg-Arg- whereas 75-80% contained the hexapeptide sequence Arg-X-Val-Phe-Arg-Arg-.