Normal Tissue Extracts Research Articles

Interaction of pRb and beta-catenin in cancer and normal tissue in the human prostate

Prostate cancer (PCa) is one of the most common oncological diseases, which goes through two stages in its development. The first stage, localized prostate cancer, can proceed indefinitely in a dormant form that does not require active medical intervention, or suddenly turn into an aggressive metastatic form with lethal outcome. The pathogenesis of the transition of the dormant form of PCa to the metastatic form remains not fully understood. The signaling pathways of the tumor suppressor pRb and the proto-oncogene β-catenin are probably the most involved in the pathogenesis of PCa but the role of their interaction in the pathogenesis of prostate cancer has not been studied. The publication on the pathogenesis of tumors in other tissues suggests that pRb may lose some properties of a tumor suppressor at the initial stage of PCa development due to its interaction with β-catenin that enables tumor cells to gain competitive advantages for reproduction. In this work, we have shown that the RB and β-catenin (CTNNB1) genes are well expressed in tumor and normal prostate tissue. Unlike β-catenin, pRb is not detected by immunoblotting in tumor and normal prostate tissue, but is easily determined in this way in extracts of control T98G cells. Co-immunoprecipitation with antibodies to pRb from extracts of tumor and normal prostate tissue makes it possible to detect this protein and β-catenin by subsequent immunoblotting, which indicates the physical interaction of these proteins in prostate tissue. On the other hand, immunoprecipitation of β-catenin with antibodies to its C-terminal fragment does not detect this protein in prostate extracts by subsequent immunoblotting using the same antibody. In contrast to prostate tissue, β-catenin is readily detected by immunoprecipitation combined with immunoblotting in T98G control cell extracts. The obtained data suggest that pRb and β-catenin physically interact with each other in cells of different tissue specificity. In T98G cells, this interaction probably occurs through the C-terminal fragment of β-catenin, but in prostate cells it occurs in a different way, since the C-fragment of β-catenin is shielded from such interaction, possibly due to its physical association with pRb.

Effect of brain traumatic tissue extract on the survival and differentiation of neural stem cells

Objective To simulate the chemical microenvironment after traumatic brain injury (TBI) and to investigate the effect of this microenvironment on the survival and differentiation of neural stem cells (NSCs). Methods The brain tissue homogenate of TBI rat model was harvested to simulate the chemical microenvironment after TBI. The primary NSCs of rat model were isolated and extraction, and then identified the phenotype characteristics with immunofluorescence staining. The experiments were divided into control group, normal brain tissue extract group (BTE group) and traumatic brain injury tissue extract group (TBITE group). The cell growth and morphological changes of each group were observed dynamically. The expression of apoptosis related protein, which includes Bax, Bcl-2, caspase-3 and cleaved caspase-3, were detected by Western Blot 24 h after experiments. The proliferation of NSCs was detected by MTT assay and Western Blot after 3 days. The differentiation level of NSCs to neurons was detected by immunofluorescence staining after 7 days. Results The results of Western Blot showed that compared with the control group, there was no significant change of apoptosis in the BTE group, while the apoptosis in the BTE group was significantly increased, showed a increase of expression levels of Bax (F=18.06, P<0.01) and Cleaved caspase-3 (F=23.86, P<0.01), and a decrease of that of Bcl-2 (F=22.95, P<0.01). The results of MTT assay showed that compared with the BTE group, the proliferation of NSCs in the TBITE group was decreased (F=41.99, P<0.01). The immunofluorescence staining showed that compared with the control group, the neuronal differentiation rate was increased in the BTE group. Further, compared with the BTE group, the neuronal differentiation rate in the TBITE group was decreased (F=66.93, P<0.01). Conclusion The injury microenvironment after TBI can significantly inhibit the survival and differentiation of NSCs, which provides a theoretical basis for clarifying the mechanism of endogenous nerve regeneration after TBI. Key words: Traumatic brain injury; Chemical microenvironment; Neural stem cells; Brain tissue extract

Role of GPR40 for Fish Oil PUFA-mediated BDNF Synthesis in the Monkey Hippocampus

Background: Polyunsaturated fatty acids (PUFA) are known to be crucial for learning and memory. However, the detailed mechanism of PUFA effects upon neuronal functions remains almost unknown except for the possible facilitation of membrane fluidity. G-protein coupled receptor 40 (GPR40) was found to induce Ca2+ mobilization in response to diverse PUFA. Thereafter, the authors found GPR40 expression in the newborn neurons of the monkey hippocampus after ischemia. This suggested implications of PUFA-mediated GPR40 signaling for adult neurogenesis underlying learning and memory. Objective: This study aims at evaluating whether PUFA-mediated GPR40 activation can affect synthesis of brain-derived neurotrophic factor (BDNF) with the aid of its proteolytic enzyme furin. Methods: Monkeys underwent 20 min transient whole brain ischemia by clamping both the innominate and left subclavian arteries. On days 7 and 15 after ischemia/reperfusion, when adult neurogenesis was shown to be maximal previously by the authors, the brain samples were resected. By the Western blotting analysis of mature-BDNF (m-BDNF), pro-BDNF and furin, syntheses of BDNF in response to two GPR40 agonists as well as selective GPR40 antagonist GW1100, were studied using normal and post-ischemic monkey dentate gyrus (DG) tissue extracts. Results: Both up-regulation of m-BDNF synthesis in response to two GPR40 agonists; fish oil PUFA and docosahexaenoic acid (DHA) and its down-regulation in response to GW1100, were observed. GPR40 antagonist inhibited m-BDNF synthesis, whereas two GPR40 agonists stimulated m-BDNF synthesis conceivably via furin activation. Cleavage of p-BDNF to m-BDNF by furin as well as syntheses of m-BDNF and furin in the DG tissues, occurred immediately after incubation with fish oil PUFA or DHA. Dynamic changes of GPR40, m-BDNF synthesis, and furin occurred simultaneously. Conclusions: These data, although correlative, suggested that m-BDNF may be synthesized by the cleavage of pre-stocked pro-BDNF and/or released from the cell store in response to PUFA. By activating GPR40 and furin, PUFA may be related to adult neurogenesis and the concomitant synaptic plasticity for learning and memory. To the best of our knowledge, this is the first report suggesting a role of GPR40 in PUFA-mediated m-BDNF synthesis.

Identification of Phospholipid Classes in Human Cancer and Normal Tissue Extracts by Nano-ESI Tandem Mass Spectrometry

Phospholipids can be classified into glycerol- and sphingo-phospholipids and into classes based on their polar head groups. The major glycerophospholipid classes are phosphatidylethanolamine (PE), phosphatidylcholine, phosphatylserine (PS) and phosphatidylinisitol (PI). In this study, total lipid and phospholipid extraction was performed according to the Bligh and Dyer method [1]. The extracts were analysed in both positive and negative ion modes ESI-MS for monitoring the present of intact phospholipids. Structural characterizations of each intact phospholipid were done in both positive and negative ion mode by electrospray ionization tandem-mass spectrometry (ESI-MS/MS). The MS/MS analyses of each protonated [M+H] + and deprotonated [M-H] molecule were performed at various different collision off set values in order to obtain the intense characteristic fragment ions in the MS/MS spectra. PC was identified in positive ion mode for all extracts due to their fixed positive charged at the choline group [2, 3] PS was able to be detected in positive and negative ion mode. In the extracts protonated PS at m/z 790 was detected at with a prominent fragment ion m/z 605. The PS species observed at m/z 790 is assigned as PS (18:0,18:1). In negative ion mode deprotonated singly charged ion and deprotonated doubly charged ion was observed at m/z 788.5 and at 393.8 respectively. PE, PI and plasmalogen-PE were also identified in the extracts. REFERENCES

Increase of N 1 ,N 12-diacetylspermine in tissues from colorectal cancer and its liver metastasis

N (1),N (12)-Diacetylspermine (DiAcSpm) is a tumor marker featured by increase in the urine of patients with cancers, including early colorectal cancer, but where and how DiAcSpm is made remains unclear. We aimed to clarify whether colorectal cancer tissues produce increased amounts of DiAcSpm, and if they do, to examine whether tissue DiAcSpm level may serve as a criterion of tissue malignancy. Tissue samples were obtained from 140 patients (13 low-grade intraepithelial neoplasia, 98 high-grade intraepithelial neoplasia and 29 colorectal cancer) treated for colorectal cancer and intraepithelial neoplasia at Tokyo Metropolitan Komagome Hospital between November 2007 and April 2011. The DiAcSpm level in cancer and adjacent normal tissue extracts was compared, and its relationship with clinical stages of the diseases was analyzed. DiAcSpm levels were higher in colorectal cancer tissue (p < 0.01, n = 12) and its liver metastasis (p < 0.05, n = 5) than in adjacent normal tissues. The tumor/normal ratio of tissue DiAcSpm content was examined for endoscopically obtained tumor and adjacent normal tissues from patients with intraepithelial neoplasia. The ratio was greater than 1.5 in 38 % (5/13) and 78 % (84/108) of low-grade intraepithelial neoplasia and high-grade intraepithelial neoplasia, respectively. Tissue DiAcSpm levels increase in the tissue of colorectal cancer and also in precancerous lesion, such as high-grade intraepithelial neoplasia. The increase is considered a sign that a tissue is acquiring malignant characteristics. It is likely that the DiAcSpm produced by cancer cells is responsible for the frequent increase in urinary DiAcSpm in early cancer patients.

Proteasome inhibition prolongs survival during lethal hemorrhagic shock in rats

Several lines of evidence suggest that proteasomes, the major nonlysosomal proteases in eukaryotes, are involved in the pathophysiology of various disease processes, including ischemia-reperfusion injury and trauma. Recently, we demonstrated that 26S proteasome activity is negatively regulated by adenosine triphosphate (ATP) and that proteasome activation during ischemia contributes to myocardial injury. The regulation of tissue proteasome activity by ATP and the potential of proteasomes as drug targets during hemorrhagic shock, however, are unknown. Thus, we evaluated the regulation of tissue proteasome peptidase activity and the effects of the proteasome inhibitor bortezomib in rat models of hemorrhagic shock. Series 1 includes animals (n = 20) hemorrhaged to a mean arterial blood pressure of 30 mm Hg for up to 45 minutes. Series 2 includes animals hemorrhaged to a mean arterial blood pressure of 30 mm Hg for 30 minutes, followed by bortezomib (0.4 mg/kg) or vehicle administration (n =5 per group) and fluid resuscitation until 75 minutes. Series 3 includes animals that underwent 40% blood volume hemorrhage, followed by 2% blood volume hemorrhage every 15 minutes until death. Bortezomib (0.4 mg/kg) or vehicle were administered 15 minutes after the onset of hemorrhage (n = 6-7 per group). Vital signs were continuously monitored. The heart, lung, and pectoral muscle were analyzed for proteasome peptidase activities and levels of ATP, ubiquitin-protein conjugates, and cytokines (tumor necrosis factor α, interleukin 6, and interleukin 10). In Series 1, proteasome peptidase activities in tissue extracts increased proportional to the decrease in tissue ATP concentrations during hemorrhagic shock. Activation of proteasome peptidase activity with decreases of the ATP assay concentration was also detectable in normal tissue extracts. In Series 2, systemic administration of bortezomib inhibited tissue proteasome activities but did not affect the physiologic response. In Series 3, bortezomib inhibited tissue proteasome activities, increased endogenous ubiquitin-protein conjugates, and prolonged survival time from treatment from 48.5 minutes in the control group to 85 minutes (p = 0.0012). Bortezomib treatment did not affect tissue cytokine levels. Proteasome activation contributes to the pathophysiology of severe hemorrhagic shock. Pharmacologic inhibition of the proteasome may provide a survival advantage during lethal hemorrhagic shock.

GST isoenzymes in matched normal and neoplastic breast tissue

The potential to metabolize endogenous and exogenous substances may influence breast cancer development and tumor growth. Therefore we investigated GST activity and the protein expression of glutathione S-transferases (GSTs) isoenzymes known to be involved in the metabolism of endogenous and exogenous carcinogens in breast cancer tissue to obtain new information on their possible role in tumor progression. The interindividual variation in the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and of 1,2-epoxy-3-(p-nitrophenoxy) propane (EPNP) with glutathione (GSH) by cytosolic glutathione S-transferases (GSTs) were investigated in human breast matched normal and tumor samples. The GSTA, GSTM, GSTP and GSTT isoenzymes from the crude extracts of matched breast normal and tumor tissues in terms of their immunological properties using western blotting were compared. In most of the samples, the GST activities were higher in the tumor than in the normal cytosolic fractions against both CDNB and EPNP. In the western blotting analysis, it was proved statistically that in normal and tumor epithelial cells, there was difference between GST pi and theta isoenzymes expressions (p0.05). In normal epithelium there was a stronger GST theta expression than in invasive tumor tissues (p=0.013). However, the stronger GST pi expression was observed in tumor epithelium than in normal epithelium in human breast cancers (p=0.000). We found the GSTP protein level and GST activities were higher in the breast tumor than in the normal cytosolic fractions against both CDNB and EPNP, thus implicating a certain biological importance.

The evolutionary atavistic endotoxin and neoplastic growth

A hypothesis on the potential role of atavistic endotoxin in carcinogenesis is proposed. The presence of an antigen identical to the endotoxin of gram-negative bacteria in tumour cells is confirmed by IgM class natural specific antibodies to endotoxin (IgMNAE) in rats by immunizing them with rat tumour tissue extracts. Rat normal tissue extracts do not increase the endogenous level of natural immunity to endotoxin, indicating the absence of a foreign antigen such as endotoxin in normal cells which are naturally devoid also of other parasitic features such as invasiveness and metastases, whereas tumour cells, during a prolonged latent period of carcinogenesis, acquire resistance to harmful factors, lose most of their genetic, antigenic, morphological and biochemical properties and become parasitic so as to survive in unfavourable conditions. With the regression of the mentioned properties of cells to the atavistic parasitic state, the synthesis of dormant endotoxin is activated together with an enhanced expression of evolutionary resistance-related genes and oncogenes. Atavistic endotoxin, produced and secreted by proliferating tumour cells, should cause chronic cachexia and septic states in cancer patients, similarly as in cases of endotoxemic septic shock where the endotoxin of gram-negative bacteria is the main pathogenic factor. Thus, the implications of the hypothesis indicate the diagnostic as well as prognostic and preventive significance of evolutionary atavistic endotoxin and also of endotoxin from gram-negative bacteria in human cancers. Natural specific antibodies to endotoxin can be helpful in creating new immunotherapeutic methods.

Parallel in Vivo and in Vitro Selection Using Phage Display Identifies Protease-dependent Tumor-targeting Peptides

We recently developed activatable cell-penetrating peptides (ACPPs) that target contrast agents to in vivo sites of matrix metalloproteinase activity, such as tumors. Here we use parallel in vivo and in vitro selection with phage display to identify novel tumor-homing ACPPs with no bias for primary sequence or target protease. Specifically, phage displaying a library of ACPPs were either injected into tumor-bearing mice, followed by isolation of cleaved phage from dissected tumor, or isolated based on selective cleavage by extracts of tumor versus normal tissue. Selected sequences were synthesized as fluorescently labeled peptides, and tumor-specific cleavage was confirmed by digestion with tissue extracts. The most efficiently cleaved peptide contained the substrate sequence RLQLKL and labeled tumors and metastases from several cancer models with up to 5-fold contrast. This uniquely identified ACPP was not cleaved by matrix metalloproteinases or various coagulation factors but was efficiently cleaved by plasmin and elastases, both of which have been shown to be aberrantly overexpressed in tumors. The identification of an ACPP that targets tumor expressed proteases without rational design highlights the value of unbiased selection schemes for the development of potential therapeutic agents.

Abstract 4566: Triplex DNA-binding proteins in resected normal and tumor tissues from colorectal cancer patients: Biological and clinical relevance

Abstract This study is to determine if triplex DNA-binding proteins in human cells play an important role in processes that lead to cancer such as genetic instability or growth deregulation. Cytoplasmic and nuclear extracts from 60 colorectal cancer patients’ tumor (T) and normal (N) tissues were isolated and examined by gel shifts (EMSA) for triplex DNA-binding proteins. Total tumor extracts showed significantly higher triplex DNA-binding activities of the major H3 complex than normal tissue extracts (Wilcoxon test). Neither T-staging nor M-staging at diagnosis was associated with any of the factors, but N-Stage was significantly associated with the ratio of tumor/normal (T/N) for the total of cytoplasmic and nuclear extracts. This means all patients without lymph node affectation had decreased binding ratios (T/N) in both cytoplasmic and nuclear extracts. Kaplan-Meier survival analysis using a cut-off of 1.5 (rounded-up median) for the nuclear binding activity ratio (T/N) showed significantly poorer survival for those patients whose nuclear binding was greater than 1.5. This suggests that although the major triplex DNA-binding protein is present in normal tissue extracts, it is more abundant in tumor extracts, and that a specific abundance of the major triplex-binding protein (H3) in the tumor nucleus is associated with poorer patient survival. Biotinylated triplex DNA-protein complexes were isolated from the RKO colorectal cancer cell line with streptavidin-agarose and analyzed by nano-HPLC ESI-MS-MS. We identified 100 and 60 kDa proteins from nuclear extracts as PSF (polypyrimidine tract binding-associated splicing factor) and p54nrb (NonO), known to function as RNA polymerase II-associated splicing factors, bind as heterodimers, and implicated in the regulation of expression of the myc family of oncoproteins and COX2. We also identified a 75 kDa protein from cytoplasmic extracts as U2AF65 (U2 small nuclear RNA auxiliary factor 2 isoform b), also a splicing factor that can directly interact with p54 nrb and shuttle continuously between the nucleus and cytoplasm. To examine differences between tumor and normal tissues in multiple signaling proteins and proteins known to be relevant in colorectal cancer, reverse phase protein array (RPPA), Pearson correlation and cluster analysis of tumor nuclear lysates ranked from lowest to highest H3 EMSA values shows a highly significant correlation between decreased β-catenin expression in tissues that have the lowest H3 EMSA values and increased β-catenin expression in tissues that have the highest H3 EMSA values. We found this pattern in all tissue lysate types: normal-cytoplasm, normal-nucleus, tumor-cytoplasm, and tumor-nucleus. We believe these splicing factors are overexpressed in tumor tissues and could cause deregulated growth of cells by influencing other proteins associated with tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4566.

Abstract 2739: Claudin-3 expression is up-regulated in epithelial ovarian cancer tissues, including tumors with minimally elevated levels of CA125

Abstract Background: Epithelial ovarian cancer (EOC) is a leading cause of death from gynecological malignancies in the US. The high mortality rate associated with EOC is largely due to the late disease stage at which the majority of women present clinical symptoms. CA125 (MUC16) is a serum marker used for detection of ovarian cancer. Serum CA125 is elevated in the majority of women with ovarian cancer, but its clinical usefulness is limited in part by low sensitivity for detection of early stage disease. Therefore, biomarkers that specify early stage ovarian cancer are needed. Objective: To identify markers that improve upon the performance of CA125, we searched for genes that were increased in tumors expressing only low levels of CA125 mRNA. We reasoned that such biomarkers may have utility in detecting the early stage ovarian cancers that are missed by screening for serum CA125 alone. Methods: The expression levels of CA125 and additional candidate biomarkers were measured using real-time quantitative RT-PCR across a panel of normal ovary tissue specimens and epithelial ovarian tumors. Biomarker protein levels in normal ovary and ovarian tumor tissue extracts were assessed by immunoblotting. Results: CA125 mRNA levels were characterized across a panel of 27 normal ovary specimens and 96 epithelial ovarian tumors. In 78% of the ovarian tumor tissues, levels of CA125 transcript were elevated greater than 10-fold relative to the average expression measured in the normal ovary specimens. In the remaining 21 EOC specimens, CA125 mRNA levels were much more modestly elevated, with increased expression measuring less than 10-fold relative to the normal ovarian tissues. Claudin-3 (CLDN3) was identified as a biomarker whose transcript level was elevated greater than ten-fold in 90% of EOC tissues including the subset of ovarian tumors expressing only modestly elevated levels of CA125 mRNA. In 17 of the 21 tumor specimens exhibiting only modestly elevated CA125 message (81%), CLDN3 transcript was up-regulated greater than 10-fold. CLDN3 message was up-regulated over 100-fold in 11 of these 21 specimens (52%). Eleven of the 21 tumors with low CA125 mRNA and high CLDN3 mRNA were diagnosed at an early stage (stage 1 + 2). This increased expression of CLDN3 mRNA in ovarian tumor tissues displaying only slightly elevated CA125 transcript was confirmed by immunoblotting tumor tissue lysates with a monoclonal antibody specific for CLDN3. Conclusion: The elevated expression of CLDN3 mRNA and protein in epithelial ovarian cancer tissues exhibiting only modest up-regulation of CA125 suggests that CLDN3 may complement CA125 as a biomarker for detection and management of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2739.

The microenvironment in chronic pancreatitis and pancreatic cancer induces neuronal plasticity

Pancreatic neuropathy in chronic pancreatitis (CP) and pancreatic cancer (PCa) is characterized by pancreatic neuropathy, i.e. increased neural density and hypertrophy, which are associated with neuropathic pain. To better understand the mechanism of these neuropathic alterations, we aimed at achieving an in-vitro simulation of the intrapancreatic neuroplasticity. Dissociated myenteric plexus (MP) and dorsal root ganglia (DRG) neurons of newborn rats were treated with normal human pancreas (NP), CP or PCa tissue extracts. Furthermore, MP and DRG neurons were cultured in supernatants from different pancreatic cancer cell lines (PCC) and human pancreatic stellate cells (hPSC) obtained from either CP or PCa tissues. For analysis, the neurite density, outgrowth, neuronal branching capacity and perikaryonal size were quantified. Myenteric plexus and DRG neurons grown in CP and PCa tissue extracts built denser networks than in NP extracts. Both neuronal types showed a strong neurite outgrowth, more complex branching pattern and a somatic hypertrophy in CP and PCa extracts. Pancreatic cancer cell supernatants induced a prominent neurite outgrowth, increased neurite density and perikaryonal hypertrophy in MP and DRG neurons. Supernatants of CP-derived hPSC strongly stimulated neurite outgrowth. Glial density in MP cultures was strikingly increased by PCa tissue extracts. Intrapancreatic microenvironment in CP and PCa induces neuroplastic alterations under in-vitro conditions, leading to increased neural density and hypertrophy. Thus, due to its neurotrophic attributes, the intrapancreatic microenviroment in CP and PCa seems to be a key player in the generation of pancreatic neuropathy and neuroplasticity.

Receptor affinity CE for measuring bioactive inflammatory cytokines in human skin biopsies

A chip-based receptor affinity CE system has been employed to measure the concentrations of bioactive pro-inflammatory cytokines in biopsy materials obtained from human atopic skin lesions. The device employs a replaceable affinity disk to which recombinant cytokine receptors have been chemically immobilized. Homogenates obtained from micro-dissected human skin samples were injected into the system where the bioactive cytokines were captured in the receptor affinity port and labeled in situ with a laser dye. The captured cytokines were released and separated by CE, the resolved peaks being detected and measured by laser-induced fluorescence. When compared with conventional cell-based bioassays, the affinity receptor chip showed reasonable correlation with r(2) values of 0.998 for interferon gamma, 0.994 for IL-6 and 0.991 for tumor necrosis factor alpha. The complete process including cytokine capture, labeling, and analysis took approximately 12.5 min with intra- and inter-assay CVs below 5.3% and recoveries of 84.9-98.4% at the 100 pg/mL concentration in buffer solutions and 84.5-95% in normal human tissue extract. The system could indicate clear differences between the various clinical stages of atopic dermatitis in human patients and could run 4-6 samples per hour. This system, like previous chip-based systems designed in our laboratory, holds the potential for being modified to be a portable unit that could be used in clinics and other biomedical screening studies.

Clinical significance of matrix metalloproteinase 2 and 9 in breast cancer

Invasion and metastasis are the most strenuous problems in the management of breast cancer. These events require diverse proteolytic enzymes, among which MMP-2 and MMP-9 play a significant role in degradation of type IV collagen, the major component of the basement membrane. Therefore, the major objective of the study is to evaluate the clinical usefulness of MMP-2 and MMP-9 with respect to malignant tumor growth, invasion, and metastasis in breast cancer. Gelatin zymography was performed on 157 tissue extracts of malignant and adjacent normal breast tissues as well as negative and positive lymph nodes from 49 breast cancer patients. Statistical analysis was carried out using SPSS statistical software (version 10). ProMMP-2 levels were significantly higher in adjacent normal tissues. Active MMP-2 and MMP-9 levels were higher in malignant breast tissues. Activation ratios of MMP-2 and MMP-9 were significantly higher in malignant breast tissues and in patients with lymph node metastasis. ProMMP-2, active MMP-2, and active MMP-9 could significantly discriminate between malignant and adjacent normal breast tissues. The MMP-2 activation ratio showed significant discriminatory efficacy between patients with and without lymph node metastasis and significant association with increased risk of lymph node metastasis in node-negative patients. The results indicate significant clinical utility of these proteolytic enzymes in malignant tumor growth, invasion, and metastasis in breast cancer.

Tissue Extracts From Infarcted Myocardium of Rats in Promoting the Differentiation of Bone Marrow Stromal Cells Into Cardiomyocyte-like Cells

To investigate whether cardiac tissue extracts from rats could mimic the cardiac microenvironment and act as a natural inducer in promoting the differentiation of bone marrow stromal cells (BMSCs) into cardiomyocytes. Three kinds of tissue extract or cell lysate [infarcted myocardial tissue extract (IMTE), normal myocardial tissue extract (NMTE) and cultured neonatal myocardial lysate (NML)] were employed to induce BMSCs into cardiomyocyte-like cells. The cells were harvested at each time point for reverse transcription-polymerase chain reaction (RT-PCR) detection, immunocytochemical analysis, and transmission electron microscopy. After a 7-day induction, BMSCs were enlarged and polygonal in morphology. Myofilaments, striated sarcomeres, Z-lines, and more mitochondia were observed under transmission electron microscope. Elevated expression levels of cardiac-specific genes and proteins were also confirmed by RT-PCR and immunocytochemistry. Moreover, IMTE showed a greater capacity of differentiating BMSCs into cardiomyocyte-like cells. Cardiac tissue extracts, especially IMTE, can effectively differentiate BMSCs into cardiomyocyte-like cells.

CYP24, the enzyme that catabolizes the antiproliferative agent vitamin D, is increased in lung cancer

1Alpha,25-dihydroxyvitamin D3 (1,25D3) displays potent antiproliferative activity in a variety of tumor model systems and is currently under investigation in clinical trials in cancer. Studies were initiated to explore its potential in nonsmall cell lung cancer (NSCLC), as effective approaches to the treatment of that disease are needed. In evaluating factors that may affect activity in NSCLC, the authors found that CYP24 (25-hydroxyvitamin D3-24-hydroxylase), the enzyme that catabolizes 1,25D3, is frequently expressed in NSCLC cell lines but not in the nontumorigenic bronchial epithelial cell line, Beas2B. CYP24 expression by RT-PCR was also detected in 10/18 primary lung tumors but in only 1/11 normal lung tissue specimens. Tumor-specific CYP24 upregulation was confirmed at the protein level via immunoblot analysis of patient-matched normal lung tissue and lung tumor extracts. Enzymatically active CYP24 is expected to desensitize NSCLC cells to 1,25D3. The authors therefore implemented a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for 1,25D3 and its CYP24-generated metabolites to determine whether NSCLC cells express active enzyme. Analysis of NSCLC cell cultures revealed time-dependent loss of 1,25D3 coincident with the appearance of CYP24-generated metabolites. MK-24(S)-S(O)(NH)-Ph-1, a specific inhibitor of CYP24, slowed the loss of 1,25D3 and increased 1,25D3 half-life. Furthermore, combination of 1,25D3 with MK-24(S)-S(O)(NH)-Ph-1 resulted in a significant decrease in the concentration of 1,25D3 required to achieve maximum growth inhibition in NSCLC cells. These data suggest that increased CYP24 expression in lung tumors restricts 1,25D3 activity and support the preclinical evaluation of CYP24 inhibitors for lung cancer treatment.

Identification of a gene on chromosome 12q22 uniquely overexpressed in chronic lymphocytic leukemia

AbstractThe pathogenesis of chronic lymphocytic leukemia (CLL) is unknown but may involve aberrant activation of signaling pathways. Somatic hypermutations in rearranged immunoglobulin heavy-chain (IgVH) genes allow a division of CLL patients into 2 categories: mutated IgVH genes are associated with an indolent disease, whereas unmutated IgVH genes define an aggressive form. Using differential display to compare gene expression in CLL cells with and without IgVH hypermutations, we identified a novel gene, CLL up-regulated gene 1 (CLLU1), that was highly up-regulated in CLL cells without IgVH hypermutations. CLLU1 mapped to chromosome 12q22, within a cluster of genes that are active in germinal center B cells. However, appreciable levels of CLLU1 were detectable only in CLL cells and not in a panel of normal tissue extracts or in any other tested hematologic malignancy. High expression of CLLU1 in CLL samples occurred irrespective of trisomy 12 or large chromosomal rearrangements. CLLU1 encodes 6 mRNAs with no sequence homology to any known gene, and most transcripts appear to be noncoding. Two transcripts, however, potentially encode a peptide with remarkable structural similarity to human interleukin 4. These data, in particular the unique and restricted expression pattern, suggest that CLLU1 is the first disease-specific gene identified in CLL.

Purification of a Human Prostate Specific Antigen.

Rabbit antiserum raised against the crude extract of normal human prostatic tissue contained antibodies to a prostatic tissue-specific antigen as shown by immunoprecipitation techniques. Using this antiserum a prostate antigen was detected in normal, benign hypertrophic, and malignant prostatic tissues, but not in other human tissues. The prostate antigen was purified to homogeneity from prostatic tissues and showed a single protein band on analytical polyacrylamide gel electrophoresis and isoelectric focusing. This report thus presents the first demonstration of the purification of a prostate-specific antigen that does not represent prostatic acid phosphatase.

Analysis of autoantibody repertoires in small- and medium-sized vessels vasculitides. Evidence for specific perturbations in polyarteritis nodosa, microscopic polyangiitis, Churg–Strauss syndrome and Wegener's granulomatosis

In order to identify new antibody reactivities, we have used a quantitative immunoblotting technique on extracts of normal human tissues to analyze the repertoires of serum IgM, serum IgG and purified IgG autoantibodies of patients with systemic vasculitides. Patients fulfilled the American College of Rheumatology and Chapel Hill criteria for the diagnosis of polyarteritis nodosa (PAN) ( n = 8), PAN related to hepatitis B virus (HBV) infection ( n = 5), Wegener's granulomatosis (WG) ( n = 6), microscopic polyangiitis (MPA) ( n = 18) or Churg–Strauss syndrome (CSS) ( n = 8). Sera from patients with chronic HBV infection without PAN ( n = 5) and age- and gender-matched healthy individuals ( n = 45) were used as controls. In the lung extract, IgM from 12/18 MPA patients reacted with high intensity with a 50 kDa band and serum IgG from 3/8 CSS patients bound to a 70 kDa protein band. In the artery extract, serum IgG from 6/18 MPA patients bound to an 85 kDa antigen, whereas purified IgG from all WG patients tested bound to a 28 kDa protein band and IgM from CSS patients bound to 2 main antigens of 38 and 60 kDa. These results provide evidence for the specificity of autoantibody repertoires from patients with PAN, WG, CSS and MPA.

Vitronectin in human breast carcinomas

We have analysed the occurrence of the extracellular glycoprotein vitronectin in carcinomas and normal tissue of human breast. Immunohistochemical analysis of carcinomas revealed a strong vitronectin accumulation in extracellular matrix (ECM) around some cancer cell clusters and in the subendothelial area of some blood vessels. In normal tissue, vitronectin had a homogeneous periductal occurrence, with local accumulation much lower than that in the carcinomas. Using a new solid phase radioligand assay, the vitronectin concentrations of extracts of carcinomas and normal breast tissue were determined and found to be indistinguishable. Comparison of the vitronectin and the hemoglobin concentrations of the extracts showed that their vitronectin content was not derived from blood contamination. Vitronectin mRNA was undetectable in both carcinomas and normal tissue. We conclude that vitronectin is not synthesised locally in breast tissue but derived by leakage from vessels, followed by extracellular accumulation in patterns distinctly different in carcinomas and normal tissue. The observation of a high vitronectin content in the carcinomas and its localisation in the tissue contributes to the clarification of the role of vitronectin in tumour biology in interaction with the plasminogen activation system and integrins.