Motility-related protein-1 (MRP-1/CD9) reduction as a factor of poor prognosis in breast cancer

The application of reliable markers is of major importance for predicting the prognosis of and instituting the appropriate postsurgical treatment of patients with breast cancer. Previously we showed that motility-related protein-1 (MRP-1), which is identical to CD9, regulates cell motility, and that cultured tumor cells transfected with MRP-1/CD9 cDNA have low motility and low metastatic potential. In addition, MRP-1/CD9 immunoblotting and immunohistochemical study with breast cancer revealed that MRP-1/CD9 expression diminished as the clinical stage of a given breast cancer advanced and that the MRP-1/CD9 gene and protein expression in the metastatic lymph nodes was strikingly lower than in the primary breast cancers. In this study, we also investigated the expression of MRP-1/CD9 by immunoblotting and immunohistochemical analysis in 143 freshly resected invasive ductal carcinomas of the breast: 52 tumors were stage I, 61 were stage II, and 30 were stage III. Tumors were classified as MRP-1/CD9 positive when a band intensity of >30% compared with positive control cells, ZR-75-30 were evaluated with the antibody M31-15, and those with intensities <30% as negative. Moreover, these results were ascertained by immunostaining. Tumor specimens classified as MRP-1/CD9 positive using Western blotting had >50% of the cancer cells immunostained with M31-15, and those classified as MRP-1/CD9 reduced had <50% of the cancer cells immunostained with M31-15. There were 97 patients with MRP-1/CD9 positive tumors and 46 patients whose tumors had reduced MRP-1/CD9 levels. The disease-free rate of the former group of patients was strikingly higher than that of the latter (84.7% versus 51.4%, P<0.001). Similarly, the overall survival rate was also significantly different between the two groups (93.6% versus 69.6%, P=0.004). Multivariate analysis with the Cox regression model indicated that MRP-1/CD9 positively correlated better with disease-free survival (P<0.001) than estrogen receptor, tumor, and lymph node status. Our data suggest that low MRP-1/CD9 expression by tumors of the breast may be associated with poor prognosis. It is conceivable that testing for MRP-1/CD9 may identify node-negative breast cancer patients who are at high risk for early disease recurrence.     

Fas/APO-1 (CD95) expression in myelodysplastic syndromes

Increased apoptosis of myeloid precursors appears to contribute to the pathophysiology of cytopenias in myelodysplastic syndromes (MDS). Fas /APO-1(CD95) is a cell surface protein inducing an apoptotic signal after its binding to Fas ligand or to a functional anti-Fas antibody. Here we studied Fas expression by immunocytochemistry on marrow slides from 30 cases of MDS. Increased Fas expression in erythroblasts and/or immature granulocytes, compared to controls, was seen in 12 (40%) of the cases. In addition, in 16 of the 18 cases with > or = 5% marrow blasts, a variable proportion of blasts expressed Fas. Increased apoptosis was found by morphological analysis and/or TUNEL technique in marrow cells from 8 of the 26 cases analyzed (31%) The ability of Fas antigen to trigger apoptosis was studied after addition of a functional anti Fas antibody in 5 of the patients with Fas overexpression. Addition of this antibody, however, only lead to mild increase of apoptosis in immature granulocytes (but not other myeloid cells) in 2 of the 5 cases. Thus, increased Fas expression is seen in myeloid and/or blast cells in the majority of MDS cases. However, the relationship between this finding and increased apoptosis in MDS still remains to be established.     

Immunohistochemically detected expression of motility-related protein-1 (MRP-1/CD9) in lung adenocarcinoma and its relation to prognosis

STUDY OBJECTIVE: To determine the effect of adding the nebulized anticholinergic drug ipratropium bromide to standard therapy compared with standard therapy alone for acute severe asthma (peak expiratory flow rate [PEFR] < 50% of predicted) in children presenting to the emergency department. METHODS: Ninety children aged 6 to 18 years were randomly assigned to two groups in a prospective, double-blind, placebo-controlled study performed in the ED of an urban children's hospital. All children received nebulized albuterol solution (.15 mg/kg) every 30 minutes, and all received oral steroids with the second dose of albuterol. Children in group 1 received ipratropium bromide (500 micrograms/dose) with the first and third dose of albuterol those in group 2 received saline placebo instead of ipratropium. Pulmonary functions (PEFR and 1-second forced expiratory volume [FEV1]) and physiologic measurements were assessed every 30 minutes up to 120 minutes. By chance, the baseline values for percent of predicted PEFR and FEV1 differed between the two groups. Therefore a multivariate model accounting for both time and baseline effects was used to compare the response between groups. RESULTS: On average, and adjusting for baseline measures, children in the ipratropium group had a significantly greater improvement in percent of predicted PEFR than did children in the placebo group at 60 minutes (P = .02), 90 minutes (P = .002), and 120 minutes (P < .0001). The improvement in percent predicted FEV1 was significantly greater for children in the ipratropium group only at 120 minutes (P = .013). Nine children (20%) from the ipratropium group and 14 (31.1%) from the control group were admitted (P = .33, chi 2). There were no significant adverse effects attributable to the ipratropium, and there was no relation between ipratropium use and changes in pulse, respiratory rate, blood pressure, or oxygen saturation. CONCLUSION: We detected significant improvement in pulmonary function studies over 120 minutes in children with severe asthma who were given nebulized ipratropium combined with albuterol and oral steroids, compared with children who received the standard therapy. Further study is needed to determine whether early use of ipratropium decreases the need for hospitalization.     

Protein kinase C regulates Fas (CD95/APO-1) expression

Fas (CD95/APO-1) is a transmembrane protein of the TNF/neuron growth factor receptor family. Ligation of Fas by specific Abs or Fas ligand (FasL/CD95 ligand) induces rapid apoptotic cell death in a variety of cell types. Despite progress in understanding the death signals transduced from Fas, very little is known with regard to the mechanisms by which Fas expression is regulated. Using our previously established murine T cell hybridoma model A1.1, we show that specific protein kinase C (PKC) inhibitors could block activation-induced Fas expression and apoptosis. The activation of PKC with PMA or 1-oleoyl-2-acetyl-sn-glycerol could mimic the TCR signal by inducing the expression of Fas but not FasL. PKC-dependent Fas expression was also observed in several murine and human tumor cell lines. Since the inhibition of Ca2+ redistribution by an inhibitor of intracellular Ca2+ mobilization, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride, inhibited TCR-induced FasL but not Fas, the expression of Fas appears to be independent of Ca2+ mobilization. Significantly, expression of the newly identified Fas-regulatory gene, TDAG51, was found to be dependent upon the activity of PKC. PKC activation only induced Fas expression in cells expressing wild-type TDAG51. Thus, Fas expression is likely mediated by PKC through TDAG51.     

Expression of human MRP6, a homologue of the multidrug resistance protein gene MRP1, in tissues and cancer cells

Arachidonic acid and eicosapentaenoic acid are important precursors for the production of prostaglandins and other hormone-like eicosanoid molecules. These fatty acids are synthesized by animals by elongating and desaturating precursor fatty acids such as linoleic acid (18:2Delta9,12) and alpha-linolenic acid (18:3Delta9, 12,15). We have identified a Delta5 fatty acid desaturase gene (fat-4) from the nematode Caenorhabditis elegans. We have expressed this gene product in Saccharomyces cerevisiae and demonstrate that it readily converts di-homo-gamma-linolenic acid (20:3Delta8,11,14) to arachidonic acid (20:4Delta5,8,11,14). The FAT-4 Delta5-desaturase also acts on a number of other substrates, including fatty acids that do not contain a double bond at the Delta8 position.     

Novel insights into structure and function of MRP8 (S100A8) and MRP14 (S100A9)

Primate lentiviruses encode for an unique nef gene with an essential function in both viral replication and pathogenicity in the host. The molecular basis for this function remains however poorly defined. Several Nef-binding cellular proteins are thought to be instrumental in its function. Indeed, Nef contains a proline-rich motif implicated in the binding to the Src-like tyrosine kinase Hck and also to a Ser/Thr kinase of molecular weight 62 kDa. The disruption of this motif affects the binding to both these kinases as well as viral replication. Whereas Hck is expressed in the myeloid lineage and hence may account for the nef function in infected monocytes, we and others have reported previously that Nef also interacts with the T-lymphocyte Src-kinase Lck, leading to specific cell signaling impairment. This interaction occurs through the binding of Nef to both Lck SH2 and SH3 domains. Both the proline motif and phosphorylation of Nef on tyrosine residue were proposed to account for these interactions. Here, we investigate the mechanism of Lck SH2 binding by HIV-1 Nef. Using recombinant fusion proteins to precipitate lysates, we show that although SH2 binding is dependent on phosphorylation events, it occurs in a tyrosine independent manner because it requires neither tyrosine residues in Nef nor the phosphotyrosine binding pocket from the Lck SH2 domain, hence suggesting a role for a phosphoserine or a phosphothreonine residue. Further, we show that Hck SH2 does not interact with Nef, indicating that Hck SH3 binding is sufficient for Nef binding, whereas Lck SH2 cooperate together with SH3 to allow Nef binding to a level similar to Hck SH3. Together, our results establish different mechanisms for Hck and Lck binding by HIV-1 Nef protein, and identify a novel mechanism for Src-like tyrosine kinase targeting by a viral protein.     

Expression of the multidrug resistance-associated protein (MRP) gene in human cancers

We determined the expression of a newly recognized drug resistance gene, the multidrug resistance-associated protein (MRP) gene, [Cole et al., Science (Washington DC), 258: 1650-1654, 1992], in normal human tissues and in >370 human tumor biopsies using a quantitative RNase protection assay and immunohistochemistry. MRP mRNA appeared to be ubiquitously expressed at low levels in all normal tissues, including peripheral blood, the endocrine glands (adrenal and thyroid), striated muscle, the lymphoreticular system (spleen and tonsil), the digestive tract (salivary gland, esophagus, liver, gall bladder, pancreas, and colon), the respiratory tract (lung), and the urogenital tract (kidney, bladder, testis, and ovary). The human cancers analyzed could be divided into three groups with regard to MRP expression. Group 1 consists of tumors that often exhibit high to very high MRP mRNA levels (e.g., chronic lymphocytic leukemia). Group 2 comprises the tumors that often exhibit low, but occasionally exhibit high MRP mRNA expression (e.g., esophagus squamous cell carcinoma, non-small cell lung cancer, and acute myelocytic leukemia). Group 3 comprises the tumors with predominantly low levels of MRP mRNA, comparable to the levels found in normal tissues (e.g., other hematological malignancies, soft tissue sarcomas, melanoma, and cancers of the prostate, breast, kidney, bladder, testis, ovary, and colon). Using the MRP-specific mAbs MRPr1 and MRPm6, we confirmed the elevated MRP mRNA levels in tumor tissues by immunohistochemistry. We conclude that hyperexpression of MRP is observed in several human cancers, and that additional studies are needed to assess the clinical relevance of MRP.     

Expression of multidrug resistance-associated protein (MRP) in brain microvessel endothelial cells

Multidrug resistance-associated protein (MRP) is a recently identified drug efflux transport system that actively transports organic acids and selected glucuronide or glutathione conjugates out of the cell. The current study presents, for the first time, both functional and biochemical data demonstrating the presence of MRP in the brain microvessel endothelial cells that form the blood-brain barrier (BBB). Using known MRP inhibitors, such as indomethacin and probenecid, fluorescein accumulation in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers was significantly enhanced compared to control. The specificity of the MRP inhibitors on cellular fluorescein accumulation was confirmed using both MRP positive (Panc-1) and MRP negative (KBv) cell lines. Furthermore, western blot analysis using a specific antibody for MRP (MRPm6) and RT-PCR studies using a complementary sequence probe for human MRP demonstrate the expression of MRP in BBMEC. Previous studies have demonstrated the significance of the P-glycoprotein drug efflux transporter in the BBB. Given its function as a drug efflux transport system, it is anticipated that MRP in the BBB will also have an important role in limiting the exposure of the brain to many endogenous and exogenous compounds, including both toxic and therapeutic agents.     

Expression of the multidrug resistance-associated protein (MRP) gene in urothelial carcinomas

The intrinsic or acquired resistance of urothelial cancer to chemotherapy is one major obstacle to successful treatment. Generally, the expression level of P-glycoprotein in urothelial cancer is low, so we accordingly investigated the expression of multidrug resistance-associated protein (MRP). We examined the expression of MRP mRNA by means of slot-blotting samples of 11 renal pelvic and/or ureteral tumors, 33 bladder tumors, one lung metastasis from a ureter tumor, 7 non-cancerous urothelia from patients with transitional-cell carcinoma (TCC) and one urothelium from a patient with renal-cell carcinoma (RCC). We also estimated, by Southern blotting, whether or not the MRP gene was amplified in clinical specimens that overexpressed MRP mRNA. MRP was detected immunohistochemically using a polyclonal antibody against MRP. In all, 5 of 11 renal pelvic and/or ureter tumors (45.5%), 17 of 33 bladder tumors (51.5%) and 4 of 7 non-cancerous urothelia of TCC patients (57.1%) expressed more than 2-fold the MRP mRNA levels of drug-sensitive human KB cells. There was no significant difference in the MRP mRNA level between primary and recurrent tumors. Low-grade urothelial carcinomas (G1 and G2 TCCs) expressed significantly higher levels of MRP mRNA than the high-grade G3 TCC. The MRP gene was not amplified in urothelial carcinomas, irrespective of their expression levels of MRP mRNA. Immunohistochemically, MRP was located mainly on the plasma membrane, but also detected on the cytoplasm of cancer cells. MRP may be one mechanism responsible for intrinsic drug resistance in low-grade urothelial cancer.     

Coordinated action of glutathione S-transferases (GSTs) and multidrug resistance protein 1 (MRP1) in antineoplastic drug detoxification. Mechanism of GST A1-1- and MRP1-associated resistance to chlorambucil in MCF7 breast carcinoma cells

To examine the role of multidrug resistance protein 1 (MRP1) and glutathione S-transferases (GSTs) in cellular resistance to antineoplastic drugs, derivatives of MCF7 breast carcinoma cells were developed that express MRP1 in combination with one of three human cytosolic isozymes of GST. Expression of MRP1 alone confers resistance to several drugs representing the multidrug resistance phenotype, drugs including doxorubicin, vincristine, etoposide, and mitoxantrone. However, co-expression with MRP1 of any of the human GST isozymes A1-1, M1-1, or P1-1 failed to augment MRP1-associated resistance to these drugs. In contrast, combined expression of MRP1 and GST A1-1 conferred approximately 4-fold resistance to the anticancer drug chlorambucil. Expression of MRP1 alone failed to confer resistance to chlorambucil, showing that the observed protection from chlorambucil cytotoxicity was absolutely dependent upon GST A1-1 protein. Moreover, using inhibitors of GST (dicumarol) or MRP1 (sulfinpyrazone), it was shown that in MCF7 cells resistance to chlorambucil requires both intact MRP1-dependent efflux pump activity and, for full protection, GST A1-1 catalytic activity. These results are the first demonstration that GST A1-1 and MRP1 can act in synergy to protect cells from the cytotoxicity of a nitrogen mustard, chlorambucil.     

Human AP endonuclease 1 (HAP1) protein expression in breast cancer correlates with lymph node status and angiogenesis

Human AP endonuclease (HAP1) plays a major role in the repair of apurinic/apyrimidinic (AP) sites in cellular DNA. We used immunohistochemistry to examine the expression of HAP1 in normal breast and in 102 primary breast carcinomas. In normal breast epithelium, HAP1 had a uniformly nuclear localization. However, in lactating glandular epithelium, the expression of HAP1 was predominantly cytoplasmic. In carcinomas, both nuclear and cytoplasmic (44%), cytoplasmic (28%) or nuclear staining (24%) were observed. In four cases (4%), no HAP1 expression was detected. All patterns of expression for HAP1 were demonstrated for ductal carcinomas in situ (DCIS), although comedo-type DCIS were usually accompanied by mostly cytoplasmic staining. Similarly, the HAP1 expression in regions of invasive tumour necrosis was cytoplasmic. Pure nuclear HAP1 expression was significantly correlated with low angiogenesis (P = 0.007) and negative lymph node status (P = 0.001). In contrast, cases with cytoplasmic as well as nuclear staining were associated with poor prognostic factors, such as high angiogenesis (P = 0.03) and node positivity (P = 0.03). The pure nuclear staining may be related to better differentiation, as in normal breast, and hence better prognostic features, and cytoplasmic staining to a more metabolically active phenotype with high protein synthesis, as in lactating breast.     

Raf-1 protein expression in human breast cancer cells

BACKGROUND: The Raf-1 kinase, a 72-kDa cytoplasmic serine-threonine kinase, plays a central role as a second messenger in signal transduction. After ligand binding to a variety of transmembrane tyrosine kinase growth factor receptors including epidermal growth factor (EGF) receptor, the 72-kDa kinase is activated through phosphorylation to a 74-kDa phosphoprotein. The Raf-1 kinase is constitutively activated in many transformed cells either directly, by mutations within its amino-terminus regulatory region, or indirectly, due to overstimulation by autocrine growth factors or activated proximal oncogenes. The role of Raf-1 kinase in breast cancer has not been studied. METHODS: To investigate the role of Raf-1 kinase expression and its activation in breast cancer, we studied three human breast cancer cell lines expressing varying amounts of EGF receptor to determine the level of Raf-1 protein and the proportion expressed in the higher molecular weight form. Effects of serum starvation and stimulation with EGF on the Raf-1 protein were studied in T47D, BT474, and MDA-MB231 cells by precipitation of cell lysates with an anti-Raf-1 antibody followed by immunoblotting. [3H]Thymidine incorporation by these cells after EGF stimulation was also determined as a measure of DNA synthesis. RESULTS: In all three breast cancer cell lines studied, the Raf-1 protein was identified in a 70- and a 74-kDa form. The level of Raf-1 was similar in all three cell lines and appeared unrelated to EGF receptor expression on the cell surface. The majority of the protein was found in the 74-kDa form even after serum starvation. A minor shift from the lower to higher molecular weight form of Raf-1 was apparent in cells treated with EGF, and increased [3H] thymidine incorporation could be demonstrated in two of the cell lines after EGF stimulation. CONCLUSION: Baseline expression of the 74-kDa or activated form of the Raf-1 kinase appeared to be elevated in the breast cancer cells studied, indicating constitutive activation. Further investigation into the role of Raf-1 protein in the pathogenesis of breast cancer is indicated.     

Activation of CD95 (APO-1/Fas) signaling by ceramide mediates cancer therapy-induced apoptosis

We report here that anticancer drugs such as doxorubicin lead to induction of the CD95 (APO-1/Fas) system of apoptosis and the cellular stress pathway which includes JNK/SAPKs. Ceramide, which accumulates in response to different types of cellular stress such as chemo- and radiotherapy, strongly induced expression of CD95-L, cleavage of caspases and apoptosis. Antisense CD95-L as well as dominant-negative FADD inhibited ceramide- and cellular stress-induced apoptosis. Fibroblasts from type A Niemann-Pick patients (NPA), genetically deficient in ceramide synthesis, failed to up-regulate CD95-L expression and to undergo apoptosis after gamma-irradiation or doxorubicin treatment. In contrast, JNK/SAPK activity was still inducible by doxorubicin in the NPA cells, suggesting that activation of JNK/SAPKs alone is not sufficient for induction of the CD95 system and apoptosis. CD95-L expression and apoptosis in NPA fibroblasts were restorable by exogenously added ceramide. In addition, NPA fibroblasts undergo apoptosis after triggering of CD95 with an agonistic antibody. These data demonstrate that ceramide links cellular stress responses induced by gamma-irradiation or anticancer drugs to the CD95 pathway of apoptosis.     

Interleukin-18/interferon-gamma-inducing factor, a novel cytokine, up-regulates ICAM-1 (CD54) expression in KG-1 cells

In a recent article (Frisbie, Forbes, and Pullum 1996) we documented racial/ethnic differences in birth outcomes according to a more fine-grained classification than has typically been employed in the demographic literature. In his commentary, van der Veen focuses on the measurement of one of the dimensions of that classification, maturity of the infant, as proxied by the fetal growth ratio. The crux of the critique is easily seen in van der Veen's statement that "all of my disagreements with Frisbie et al.'s method arise from their particular use of a postnatal standard for the assessment of intrauterine growth." Our critic misunderstands our objective: He fails to realize our interest in birth outcome, not pregnancy process, and does not perceive that our intent was to extend the research extant in both the demographic and public-health literatures in which patently postnatal (i.e., ex utero) measures are taken as outcomes interesting in their own right and/or as risk factors for infant mortality and infant and childhood morbidity. Specifically, he does recognize that we purposefully expanded our focus to include moderately compromised births to determine if they were at higher risk than the normal births with whom they are conventionally categorized. Our discussion draws on research cited in the original article, on studies cited by our critic, and on a few more recent investigations. Although we have never argued that ours is the only, or even the best approach in all cases, we try to clarify the rationale for, and adduce additional empirical evidence of, the utility of the method we used.