T cells reactive with HLA-A*0201 peptides from the histone demethylase JARID1B are found in the circulation of breast cancer patients

The nuclear protein PLU-1/JARID1B/KDM5 is widely expressed in breast cancers while showing highly restricted expression in normal adult tissues. To investigate whether JARID1B is a potential target antigen for immunotherapy of breast cancer, we have analyzed the responses of CD8(+) T cells to JARID1B HLA-A*0201 peptides in vitro and used peptide multimers to detect the presence of JARID1B reactive T cells in the circulation of breast cancer patients. Peptides were selected using two web-based algorithms: criteria for inclusion being a high score in both prediction algorithms, and nonhomology with retinoblastoma binding protein-2 (RBP2/JARID1A/KDM5A). A 65-peptide panel was selected and assayed for binding strength by competition assay to obtain the IC(50). The immunogenicity in vitro of these peptides was assessed by T cell stimulation experiments, using autologous dendritic cells as APCs in the first rounds followed by autologous lymphoblasts. Fourteen of the peptides assayed produced cultures having >2% of the CD8(+) cells being IFN-γ(+) after 3-6 rounds of stimulation. An HLA-A*0201 cell line could activate the specific T cells if pulsed with peptide, but endogenous peptide levels were insufficient for activation. Nevertheless, multimer staining of circulating T cells from breast cancer patients showed a significantly higher percentage of multimer positive CD8(+) T cells, as compared to healthy adults for two of three JARID1B epitopes tested. One of these, peptide 73 (QLYALPCVL), was analyzed for memory phenotype, and found to have a significantly higher proportion of central memory T cells than the control group, demonstrating a previous exposure to the peptide.     

Serological discrimination between HTLV-I and HTLV-II antibodies by ELISA using synthetic peptides as antigens

Using the peptides from amino acids 100-130 of the HTLV-I gag protein, 175-199 of the HTLV-I env protein and the corresponding peptides of HTLV-II (amino acids 106 to 135 of the gag protein and 171 to 196 of the env protein), we tested for reactivity against antibodies by enzyme immunoassay in sera from HTLV-I and HTLV-II carriers. The peptides derived from the env proteins have high specificity for antibody binding. The peptide based on amino acids 175-199 of HTLV-I reacted with antibodies in sera from all HTLV-I carriers, and the peptide composed of amino acids 171-196 of HTLV-II reacted with antibodies in sera from all HTLV-II carriers. For the peptides derived from the gag proteins, we observed some cross-reactivity in sera from persons with anti-HTLV-I and anti-HTLV-II, due to antibody binding to the peptide corresponding to 12 amino acids from the C-terminal end of the gag protein. Separate enzyme immunoassays that used the four synthetic peptides as antigens clearly distinguished between serum with antibodies to HTLV-I or HTLV-II in various individuals and excluded false positive results using the particle agglutination assay that used a whole-virus lysate of HTLV-I as antigen.     

Preferential induction of necrosis in human breast cancer cells by a p53 peptide derived from the MDM2 binding site

p53 is the most frequently altered gene in human cancer and therefore represents an ideal target for cancer therapy. Several amino terminal p53-derived synthetic peptides were tested for their antiproliferative effects on breast cancer cell lines MDA-MB-468 (mutant p53), MCF-7 (overexpressed wild-type p53), and MDA-MB-157 (null p53). p53(15)Ant peptide representing the majority of the mouse double minute clone 2 binding site on p53 (amino acids 12-26) fused to the Drosophila carrier protein Antennapedia was the most effective. p53(15)Ant peptide induced rapid, nonapoptotic cell death resembling necrosis in all breast cancer cells; however, minimal cytotoxicity was observed in the nonmalignant breast epithelial cells MCF-10-2A and MCF-10F. Bioinformatic/biophysical analysis utilizing hydrophobic moment and secondary structure predictions as well as circular dichroism spectroscopy revealed an alpha-helical hydrophobic peptide structure with membrane disruptive potential. Based on these findings, p53(15)Ant peptide may be a novel peptide cancer therapeutic because it induces necrotic cell death and not apoptosis, which is uncommon in traditional cancer therapy.     

Inhibition of proteinase-like peptidase activities in serum and tissue from breast cancer patients

The inhibitory profiles of several proteinase-like peptidases active on synthetic peptide (MCA) substrates, present in sera and 100,000g supernatants of malignant tissue from patients with breast cancer, have been studied using a series of known inhibitors including epoxysuccinyl peptides (E-64, Ep-475), Z-Phe-Phe-diazomethane, PMSF, iodoacetamide, 1-10-O-phenanthroline, leupeptin, aprotinin, elastatinal and alpha 2-macroglobulin. While in general the inhibition profiles confirmed reported substrate specificities some anomalies were observed. In particular, the serum activities on two cathepsin B substrates were unaffected by specific cysteine proteinase inhibitors and in breast tissue only 20-37% of activity towards these two substrates was apparently due to the presence of endopeptidases. However, the potent inhibition of other proteinase-like activities by the epoxysuccinyl peptides and leupeptin, or similar inhibitors, may be useful agents in the study of methods of combating tumour spread.     

Synthetic peptides reactive with anti-human milk fat globule membrane monoclonal antibodies

Mammary mucins are increased in amounts in breast cancer patient sera, and most anti-breast cancer antibodies react with such mucins. One such mucin is found in human milk fat globule membrane and consists predominantly of O-linked sugars and a protein core. Partial complementary DNA clones for the protein core have recently been obtained. The nucleotide sequence is of interest as it contains a 60-base pair repeat, giving rise to a repeated 20-amino acid sequence (PDTRPAPGSTAPPAHGVTSA). Peptides with various lengths were synthesized using this sequence and the adjacent 4 amino acids (PDTR). Three anti-human milk fat globule membrane antibodies produced in our laboratory (BC1, BC2, and BC3) were tested to determine their reactivity with these synthetic peptides. Using three different assays (direct enzyme-linked immunosorbent assay test on peptides, direct enzyme-linked immunosorbent assay test on bovine serum albumin-conjugated peptides, and an inhibition test with the peptides in liquid, rather than solid phase), it was shown that APDTR was the minimum amino acid sequence required to form a reactive epitope with all 3 antibodies, although individual differences in the reactivities of the antibodies were noted. The addition of alanine (A) converted a nonreactive PDTR peptide to a reactive one, and the deletion of arginine (R) did the reverse; thus APDTR is the smallest peptide which reacts with these anti-human milk fat globule membrane antibodies.     

Detection of bladder cancer using a novel nuclear matrix protein, BLCA-4.

We have identified previously six nuclear matrix proteins (NMPs) that are bladder cancer specific. In this study, we analyzed the expression of one of these proteins, BLCA-4, in bladder tumors and normal bladder tissue. We also examined the appearance of BLCA-4 in the urine as a biomarker for bladder cancer. BLCA-4 was isolated from nuclear matrix preparations of bladder tumors, and its peptide sequence was determined. The antibodies generated against the resulting BLCA-4 peptides were then used to detect its presence in immunoblots and in urine samples by immunoassay. We analyzed tissue samples of bladder tumor and normal donor bladders and urine obtained from 51 normal individuals and 54 patients with pathologically confirmed bladder cancer. The BLCA-4 peptide sequences do not resemble any known human protein sequences. On immunoblot analysis, BLCA-4 expression was detectable in tumor and normal tissues from patients with bladder cancer but not in any of the normal bladder tissue obtained from organ donors. Using a prospectively determined cutoff level of 13 A (absorbance) units/microg protein, all 51 normal individuals tested were negative for BLCA-4 expression, whereas 53 of 55 samples from patients with bladder cancer were positive. These results suggest that BLCA-4 is present throughout the bladder in both the tumor and morphologically normal areas in bladder cancer patients. BLCA-4 is a very sensitive (96.4%) and specific (100%) marker for bladder cancer. BLCA-4 is a bladder cancer-specific marker that can be detected using a urine-based assay and can be used in the diagnosis of bladder cancer.     

C1B domain peptide of protein kinase Cγ significantly suppresses growth of human colon cancer cells in vitro and in an in vivo mouse xenograft model through induction of cell cycle arrest and apoptosis

Two peptides derived from the C1B domain of protein kinase Cγ (PKCγ) were shown to associate with classical PKC isozymes and modulate their activities. These C1B peptides are designated C1B1 (amino acid residues 101-112) and C1B5 (residues 141-151). Since PKC enzyme activity is shown to be involved in colon cancer development, the effect of C1B peptides on the growth of various human colon cancer cell lines was examined in vitro and in vivo. Sub-micromolar to micromolar levels of both C1B peptides induced approximately 60-70% growth attenuation in multiple colon cancer cell lines in a soft agar tumor colony assay; however, C1B5 peptide was not cytotoxic to normal colon epithelial cells in two dimensional culture. The effect of C1B5 peptide on colony growth of COLO205 cells was reversed by treatment with the PKCα/β inhibitor, Ro-32-0432. C1B peptide treatment attenuated COLO205 cells via two mechanisms: 1) cell cycle arrest and 2) stimulation of apoptosis. This is evident in G 2 arrest and increases in levels of cleaved caspase 3 and p53 phosphorylated at serine 20. Intratumoral injection of C1B5 peptide (20 mg/kg/day, every three days) markedly attenuated the growth of subcutaneous xenografts of COLO205 cells in SCID mice by 76% compared with the control. Taken together, these results strongly suggest that C1B peptides have negligible effects on normal tissues but are potentially effective chemotherapeutic agents for colon cancer.     

瘦素及瘦素受体与乳腺癌浸润、转移关系的研究

目的：了解瘦素及瘦素受体与乳腺癌浸润、转移关系。方法测量对比良性乳腺疾病与乳腺癌患者体重指数（body mass index,BMI）的差别;按腋窝淋巴结转移数由低到高分5组,S-P 方法检测不同分组中乳腺癌组织中瘦素及其受体的表达,ELASE 方法检测乳腺癌患者血清中瘦素的表达。结果良性乳腺疾病患者 BMI 为24．36±3．74,乳腺癌患者 BMI 为25．77±4．12,二者比较P＝0．028。瘦素在乳腺癌组织中的表达为：A 组163．87±16．33,B 组147．24±8．03,C 组133．54±18．82,D 组105．73±10．39,E 组81．52±8．72;乳腺癌组织中瘦素受体的表达为：A 组147．93±12．31,B 组125．03±16．15,C 组113．85±17．59,D 组107．25±12．85,E 组80．47±7．38;乳腺癌血清中瘦素的表达为：A组16．378±0．471,B 组21．393±1．551,C 组28．978±2．570,D 组35．706±2．942,E 组39．186±7．377。结论肥胖是乳腺癌发病的高危因素。瘦素及其受体在乳腺癌组织中的表达随淋巴结转移数增多而依次升高,提示瘦素及其受体促进乳腺癌的转移、复发。     

Cancer-associated SCM-recognition, immunedefense suppression, and serine protease protection peptide. Part I. Isolation, amino acid sequence, homology, and origin.

The isolation of a cancer-associated, SCM-recognition, immunedefense-suppressing, and serine protease-protecting (CRISPP) peptide from the blood plasma of cancer patients is described. The amino acid sequences were determined on preparations from 12 different cancers. The peptide is composed in 9 cancers of 29 and in 3 cancers of 35 amino acid residues with molecular weights of 3410 and 4007 Da, respectively. A consensus, synthetic 29 amino acid CRISPP peptide (CRISPPs) has the same cancer SCM-recognition (CR) activity and SCM-response modifying effects as the natural peptide. The "cancer SCM-recognition epitope" of the CRISPP peptide was determined. Anti-CRISPPs antibodies were raised and used in immunoassays to confirm the presence of the CRISPP peptides in cancer blood plasmas, in supernatants of cancer cell growth media and in cultured human cancer cells. The amino terminal end sequences of peptides isolated from growth media of cultured breast and colon cancer cells corresponded to amino acid sequences of CRISPP peptides isolated from cancer blood plasmas of subjects with the respective cancers. The CRISPP peptides are between 83 to 100% homologous to the alpha 1-protease inhibitor amino acid sequence located at the carboxy terminal end between residues 358 and 393. The genetic origin of the CRISPP peptides and their selective advantage to cancer cell survival are discussed.     

Context of MUC1 epitope: immunogenicity

MUC1 is a glycoprotein found at the secretory poles of normal cells but is hypoglycosylated on the entire surface of cell membranes of adenocarcinomas. In order to determine the influence on the immune response of peptide context for epitope presentation, peripheral blood mononuclear cells (PBMC) from patients with adenocarcinomas, were stimulated with MUC1 peptides derived from the 20 amino acids (aa) long sequence that is characteristic of the MUC1 Variable Number of Tandem Repeats (VNTR). In the seven peptides tested, the T-cell tumor-specific epitope (cTSE) was surrounded by variable numbers of aa and repeated up to 5 times in the same peptide. The results of this study indicate that cultures stimulated with peptide 610 (GSTAPPAHGVTS APDTRPAP) showed the highest specific killing of the MUC1-expressing breast cancer MCF-7 cells. Peptide 610 is also superior to the other peptides in inducing better production of the type 1 cytokines, tissue necrosis factor alpha and interferon gamma. In conclusion, context of the epitope and not sequence alone determines immunogenicity.     

Phase-I study of synthetic muc1 peptides in breast-cancer

Exposed peptides in the repeat (VNTR) protein core of human mucin 1 (MUC1) could be a target for immunotherapy, as it is highly immunogenic in mice and a human cytotoxic T lymphocytes to MUC1 recognise the peptide. On this basis 13 patients were immunised with a MUC1 peptide - a 20 amino acids dimer conjugated with diphtheria toroid as carrier. In patients with established breast cancer increasing doses (0.15 mg, 0.25 mg, 0.5 mg, 1.0 mg) were used at 2 week intervals (3 injections). No toxicity was found, other than for DTH reaction to the diphtheria carrier; weak antibody and T cell proliferative responses were seen and weak DTH reaction in proportion of patients. The MUC1 peptide appears to be safe but in the form used was not highly immunogenic.     

C-erbB-2 protein in the sera of breast cancer patients

Summary The c-erbB-2 protein was measured in sera of patients with breast cancer or benign breast diseases to study the significance of this protein as a tumor marker. The mean value and positive rate for this protein (assuming 20 U/ml as the cut-off value) were 11.8 U/ml (0%) in benign breast disease (n=30), 11.8 U/ml (3.1%) in stage I/II primary breast cancer (n=64), 38.2 U/ml (29.4%) in stage III/IV primary breast cancer (n=17), 17.9 U/ml (33.3%) in locally recurrent breast cancer (n=12), 298.4 U/ml (51.0%) in recurrent breast cancer with distant metastases (n=51), and 12.9 U/ml (0%) in those with no evidence of recurrence (n=57). Thus, the serum c-erbB-2 protein level was significantly higher in the distant metastatic group. In patients with distant metastases, there was a close association between expression of c-erbB-2 protein in the primary tumor and the serum c-erbB-2 protein level. On the basis of these results, serum c-erbB-2 protein was thought to be useful as a tumor marker for postoperative monitoring of breast cancer, especially in patients positive for expression of this protein in primary cancer tissue.     

Breast cancer growth inhibition by delivery of the MDGI-derived peptide P108

Mammary derived growth inhibitor (MDGI) is a member of the family of cytoplasmic fatty acid binding proteins (FABPs), which bind hydrophobic ligands such as fatty acids, retinoids, eicosanoids and prostaglandines. MDGI and an 11 amino acid MDGI-derived conserved C-terminal peptide (P108) inhibits growth of normal mammary epithelial cells in tissue and organ culture, but fails to inhibit proliferation of many breast cancer cell lines in vitro. Here, the effects of peptide P108 on tumor growth of MCF-7, MDA-MB468 and MDA-MB231 human breast cancer cell lines in nude mice were tested. To deliver P108 into tumors, a novel peptide production system was applied for expression and secretion of small bioactive peptides in mammalian cells. Functional differentiation was observed in MCF-7 and MDA-MB468 cells upon P108 expression. In addition, EGF-dependent colony formation in soft agar by MDA-MB468 cells was inhibited by secreted P108. Tumor growth in athymic nude mice was suppressed in all three cell lines tested. Furthermore, P108 expressed by MCF-7/P108 cells caused paracrine tumor growth inhibition of MDA-MB231 cells. These results indicate that breast cancer inhibition by P108 is independent of binding to hydrophobic ligands and is perhaps mediated by interference with EGF-dependent signaling pathways.     

Comparison between p53 staining in tissue sections and p53 proteins levels measured by an ELISA technique.

We studied 51 paired samples of tissue sections and cytosol extracts from patients with breast cancer. A very high affinity monoclonal antibody to human p53 protein, DO-1, and polyclonal serum CM-1 to p53 protein were used for two site ELISA assays and CM-1 was used for immunohistochemistry to detect p53 protein accumulation in breast cancer samples. Eighteen carcinomas were positive for p53 by tissue staining and ELISA assay. Nineteen tumours were negative by ELISA and immunohistochemistry, and 14 cases with low levels of positive staining by immunohistochemistry were negative by the ELISA assay. A statistically significant correlation has been found between the degree of staining and the amount of p53 protein measured by ELISA (Pearson''s correlation coefficient r = 0.59, P < 0.00001). Our ELISA assay offers an alternative approach to evaluating the p53 status of breast biopsy material, using cytosol extracts routinely prepared for steroid hormone receptor assays. This assay should also be of general application to other situations where the level of p53 protein needs to be determined.     

Nuclear targeting of a midregion PTHrP fragment is necessary for stimulating growth in breast cancer cells

Parathyroid-hormone related protein (PTHrP) is the primary factor in humoral hypercalcemia of malignancy and is highly secreted by breast cancers. The pro-hormone undergoes post-translational processing and cleavage to give rise to mature secretory peptides, one of which is midregion PTHrP (38-94/95/101) containing a nuclear localisation sequence (NLS) in amino acids (87-106). The current study investigates whether the NLS in midregion PTHrP is important in breast cancer growth. PTHrP-(67-101), a midregion PTHrP fragment containing NLS-(87-101) significantly increased growth of MCF-7 and MDA-MB231 cells (126.3 and 121.3% of control respectively in serum conditions), independent of PTHR1 whereas PTHrP-(67-86), which lacks the NLS did not. Fluorescent-labelled PTHrP-(67-101) translocated to the nucleus, whereas PTHrP-(67-86) remained cytosolic and a scrambled(+NLS) peptide was not internalised. In comparison, no growth influence or uptake was seen in non-tumour breast cells (Hs578Bst). Increases in intracellular calcium mobilisation were observed in breast cancer cells stimulated with both PTHrP-(67-101) and PTHrP-(67-86) (EC(50) of 3.2 pM and 2.2 pM respectively for MCF-7 cells), whereas inositide turnover was not detected. Both nuclear uptake and calcium signalling were attenuated in the presence of EGTA, but not with U73122 or N-terminal PTHrP peptides. Our studies indicate that the NLS-containing midregion PTHrP peptide is dependent on both internalisation and nuclear translocation to induce growth in breast cancer cells. These findings highlight the importance of midregion PTHrP and its receptor in breast cancer growth and may provide potential targets for future therapeutic intervention.     

基于超高效串联质谱的氨基酸代谢组学预测晚期乳腺癌化疗反应初探

  目的  探讨晚期乳腺癌患者化疗前(基线)和化疗第1个疗程后(预后)血清中32种氨基酸的变化,预测其指导晚期乳腺癌化疗敏感性的价值。  方法  选取2015年3月至2016年10月于辽宁省肿瘤医院行化疗的女性晚期乳腺癌患者73例,分别在化疗前和化疗第1个疗程后采集外周血2 mL,经超高效串联质谱(LC-MS/MS)检测血清中32种氨基酸的含量水平。采用影像学检查对化疗2~4个疗程后患者行预后评估,分为好转组和恶化组,并分析氨基酸含量水平的变化。  结果  32种氨基酸含量水平为3~180 000 pmol/L。与基线相比,好转组的甘氨酸和L-谷氨酰胺相对含量显著上升,而恶化组显著下降;好转组的肌氨酸显著下降,而恶化组变化不明显;好转组和恶化组的L-苏氨酸、牛磺酸、亚氨基二乙酸和L-谷氨酸均显著上升。  结论  甘氨酸和肌氨酸等氨基酸在行化疗前和化疗第1个疗程后的变化对晚期乳腺癌化疗疗效具有一定的预测价值,较影像学检查可能更早预测化疗疗效,有助于指导患者的治疗。关键词晚期乳腺癌氨基酸代谢化疗敏感性超高效串联质谱      

A new monoclonal antibody, P2A8(6), that specifically recognizes a novel epitope on the multidrug resistance-associated protein 1 (MRP1), but not on MRP2 nor MRP3

Multidrug resistance (MDR) is a major problem in the chemotherapeutic treatment of cancer. Overexpression of the multidrug resistance-associated protein 1 (MRP1), is associated with MDR in certain tumors. A number of MRP1-specific MAbs, which facilitate both clinical and experimental investigations of this protein, are available. To add to this panel of existing antibodies, we have now generated an additional MRP1-specific monoclonal antibody (MAb), P2A8(6), which detects a unique heat stable epitope on the MRP1 molecule. Female Wistar rats were immunized via footpad injections with a combination of two short synthetic peptides corresponding to amino acids 235-246 (peptide A) and 246-260 (peptide B) of the MRP1 protein. Immune reactive B cells were then isolated from the popliteal lymph nodes for fusion with SP2/O-Ag14 myeloma cells. Resultant hybridoma supernatants were screened for MRP1-specific antibody production. Antibody P2A8(6) was characterized by Western blotting and immunocytochemistry on paired multidrug resistant (MRP1 overexpressing) and sensitive parental cell lines. The antibody detects a protein of 190 kDa in MRP1-expressing cell lines but not in MRP2- or MRP3-transfected cell lines. P2A8(6) stains drug-selected and MRP1-transfected cell lines homogeneously by immunocytochemistry and recognizes MRP1 by immunohistochemistry on formalin-fixed paraffin wax-embedded tissue sections. Peptide inhibition studies confirm that P2A8(6) reacts with peptide B (amino acids 246-260), therefore recognizing a different epitope from that of all currently available MRP1 MAbs. This new MAb, chosen for its specificity to the MRP1 protein, may be a useful addition to the currently available range of MRP1-specific MAbs.