Mammaglobin as a potential molecular target for breast cancer drug delivery

Background

Mammaglobin (MAM) has been used as a specific molecular marker for breast cancer diagnosis. Recently, several groups of researchers proposed a number of therapeutic strategies targeting this molecule. Some of the strategies are based upon an essential but not demonstrated hypothesis – mammaglobin is associated with the surface of breast cancer cells, which strongly disputes the therapeutic strategies.

Results

We conducted a computer-based predictive analysis and identified a small fragment at the N-end of MAM as a potential transmembrane domain. We provided several evidences to demonstrate the presence of the membrane-associated MAM. We isolated the membrane protein components from known MAM positive breast cancer cells (MDA-MB361 and MDA-MB415). We showed that about 22–64% of MAM proteins, depending upon the types of the cancer cells, directly attached on the membrane of breast cancer cells, by Western blotting assays. To directly visualize the presence of the membrane-bound MAM protein, we incubated the MAM positive cancer cells with FITC labeled anti-MAM antibody, and observed clear fluorescent signals on the surface of the cells. In studying the MAM protein distribution in human breast cancer tissues, we first identified two immunostain patterns that are associated with the membrane-bound MAM: the membrane stain pattern and luminary surface stain pattern. To test whether the membrane-associated MAM can serve as a molecular target for drug delivery, we conjugated anti-MAM antibody to human low-density lipoprotein (LDL) and loaded doxorubicin (Dox) in the core of LDL. Specific binding and cytotoxicity of the MAM targeted and Dox loaded LDL was tested in the MAM positive breast cancer cells in vitro.

Conclusion

We first showed that some of MAM protein directly associated with the surface of breast cancer cells. The membrane-associated MAM protein may be utilized as a useful molecular marker for breast cancer targeted drug delivery.     

Synapsin-1 is found in a microtubule-associated complex of proteins isolated from bovine brain

We have prepared microtubules from brain tissue by stabilizing the cellular microtubules in 6.7 M glycerol buffer, instead of the usual procedure which extracts the solubilized protein and then reassembles microtubules in vitro at some later time. There are substantial differences in the microtubule associated proteins obtained by the two methods, and brain spectrin is a major component of the stabilized microtubules. We have now modified the buffer used for the isolation of stabilized microtubules to minimize their tendency to aggregate. When the stabilized microtubules were further purified by sucrose density gradient centrifugation, we were able to distinguish previously unidentified polypeptides at 49, 74 (doublet), and 100 kilodaltons (doublet). These bands maintained staining intensity in the same proportion to tubulin as in the original homogenate, whereas background proteins were diminished in staining intensity. We now report the identification of the 74-kilodalton doublet polypeptides as synapsin-1 by peptide mapping. Synapsin-1 is a protein known to bind to brain spectrin and also to microtubules, and may thus serve as a linker between these cytoskeletal components.     

Estrogen receptor gene amplification is found in some estrogen receptor-positive human breast tumors

The genomic organization of the estrogen receptor (ER) gene has been analyzed in 21 primary human breast cancers and 1 axillary metastasis. No evidence of rearrangements of the ER gene was found in the analyzed tumors. In 6/14 ER-positive tumors a certain degree of amplification of the ER gene, ranging from 1.6 to 3-fold, was detected. No correlation was observed between the level of gene amplification and the amount of ER in the tumors. In the 8 ER-negative tumors analyzed no amplification could be detected. It is concluded that ER gene amplification may be one of the mechanisms underlying the increased ER expression in some breast tumors.     

DJ-1 is present in a large molecular complex in human brain tissue and interacts with alpha-synuclein

DJ-1 is a ubiquitously expressed protein involved in various cellular processes including cell proliferation, RNA-binding, and oxidative stress. Mutations that result in loss of DJ-1 function lead to early onset parkinsonism in humans, and DJ-1 protein is present in pathological lesions of several tauopathies and synucleinopathies. In order to further investigate the role of DJ-1 in human neurodegenerative disease, we have generated novel polyclonal and monoclonal antibodies to human DJ-1 protein. We have characterized these antibodies and confirmed the pathological co-localization of DJ-1 with other neurodegenerative disease-associated proteins, as well as the decrease in DJ-1 solubility in disease tissue. In addition, we report the presence of DJ-1 in a large molecular complex (> 2000 kDa), and provide evidence for an interaction between endogenous DJ-1 and alpha-synuclein in normal and diseased tissue. These findings provide new avenues towards the study of DJ-1 function and how loss of its activity may lead to parkinsonism. Furthermore, our results provide further evidence for the interplay between neurodegenerative disease-associated proteins.     

Proteasome subunit zeta,a putative ribonuclease,is also found as a free monomer

20 S Proteasomes are large proteinase complexes found in eukaryotic cells where they degrade cell proteins in an ATP-dependent manner. Proteasomes consist of 14 different subunits. One of them, zeta, was found in HeLa cells at a concentration of 890 g per g of cell protein. A large proportion of zeta was found in the free state rather than incorporated into proteasomes, namely 28% in HeLa cells and 37% in BSC-1 cells. Free zeta was found in both nuclei and cytoplasm. In HeLa cells free zeta had a t1/2 of 2.8 h, compared to 5 d for proteasomes, and did not exchange with zeta in proteasomes. We confirmed (Petit F et al.: Biochem. J. 326: 93–98 (1997)) that both 20 S proteasomes and free zeta subunits possess RNase activity though the activities were very low: 4 mMoles and 0.6 mMoles of tobacco mosaic virus RNA degraded per mole of enzyme per min, respectively. The physiological function of the relatively abundant zeta monomers is not known.     

TEX101, a germ cell-marker glycoprotein, is associated with lymphocyte antigen 6 complex locus k within the mouse testis

In adult male mice, the glycosylphosphatidyl inositol-anchored glycoprotein TEX101 is expressed only in germ cells and is thought to be involved in spermatogenesis. However, the details regarding the function of TEX101 remain to be clarified. We previously identified Ly6k as a candidate TEX101-associated protein, but as molecular probes are not currently available to detect Ly6k, we do not have conclusive evidence of the association between TEX101 and Ly6k. In this study, we confirmed the biological interaction between TEX101 and Ly6k using an established anti-mouse Ly6k polyclonal antibody (pAb). A combination of immunoprecipitation, Western blot, and immunohistochemical analyses using the pAb revealed that TEX101 is physically associated with Ly6k within the testis. In addition, these proteins simultaneously co-migrate into the detergent-resistant membrane fractions, suggesting that TEX101 collaborates with Ly6k on the cell membrane and may play a role in spermatogenesis.     

Ferritin mRNA is found on bound as well as on free polyribosomes in rat heart

Free and endoplasmic reticulum-bound polyribosomes from rat heart were examined for their ferritin mRNA content. A procedure for separation and purification of the two ribosome populations that produced good yields of homogeneous mono- and polyribosomes with no contaminating ultrastructures and gave distinctive sedimentation profiles in 15-50% sucrose gradients was developed. 14C-labeled free and bound polyribosomes added to heart preparations indicated that only 3% of free and 5.5% of bound polyribosomes cross-contaminated the bound and free fractions, respectively. RNA from both polyribosome populations hybridized with [32P]cDNA for rat ferritin. The extent of hybridization with mRNA from endoplasmic reticulum (ER)-derived polyribosomes was much greater than what could be accounted for by cross-contamination with free polyribosomes. This indicates that heart ferritin is synthesized not only on free polyribosomes for internal use in iron storage but also on ER-bound polyribosomes, where it may be destined for secretion into the plasma.     

Some observations on a protein-mucopolysaccharide complex found in sea urchin embryos

A complex of protein and mucopolysaccharide was isolated chromatographically from developing sea urchin embryos. The protein moiety of the complex was found to vary electrophoretically from one developmental stage to another. On the other hand, the mucopolysaccharide moiety was invariable throughout all the stages tested, and was very probably heparitin sulfate. The results of pulse-labeling and chase experiments showed that the complex was synthesized in the cytoplasm and subsequently transferred into the nucleus. Data were obtained which indicate the possibility that the mucopolysaccharide in the nucleus is localized preferentially in the template-active part of the chromatin. An augmentation of RNA synthesis in isolated nuclei was observed when the complex was added to the incubation mixture. A possible role of the complex molecule in the mechanisms of nucleo-cytoplasmic interactions is discussed.     

SAG101 forms a ternary complex with EDS1 and PAD4 and is required for resistance signaling against turnip crinkle virus

EDS1, PAD4, and SAG101 are common regulators of plant immunity against many pathogens. EDS1 interacts with both PAD4 and SAG101 but direct interaction between PAD4 and SAG101 has not been detected, leading to the suggestion that the EDS1-PAD4 and EDS1-SAG101 complexes are distinct. We show that EDS1, PAD4, and SAG101 are present in a single complex in planta. While this complex is preferentially nuclear localized, it can be redirected to the cytoplasm in the presence of an extranuclear form of EDS1. PAD4 and SAG101 can in turn, regulate the subcellular localization of EDS1. We also show that the Arabidopsis genome encodes two functionally redundant isoforms of EDS1, either of which can form ternary complexes with PAD4 and SAG101. Simultaneous mutations in both EDS1 isoforms are essential to abrogate resistance (R) protein-mediated defense against turnip crinkle virus (TCV) as well as avrRps4 expressing Pseudomonas syringae. Interestingly, unlike its function as a PAD4 substitute in bacterial resistance, SAG101 is required for R-mediated resistance to TCV, thus implicating a role for the ternary complex in this defense response. However, only EDS1 is required for HRT-mediated HR to TCV, while only PAD4 is required for SA-dependent induction of HRT. Together, these results suggest that EDS1, PAD4 and SAG101 also perform independent functions in HRT-mediated resistance.     

Aggressiveness of breast cancers found with and without screening.

OBJECTIVE--To examine how breast cancers found by mammographic screening differ from those found outside screening. DESIGN--Comparative cohort study. SETTING--Turku, southwestern Finland. PATIENTS--126 women aged 40-74 years with breast cancer detected during the first round of mammographic screening in 1987-90 and 125 women within the same age range with breast cancer detected outside screening during the same period. MAIN OUTCOME MEASURES--Primary tumour size, axillary nodal status, histological features, oestrogen and progesterone receptor concentrations, ploidy, and S phase fraction. RESULTS--Compared with the controls women with cancers detected by screening had a smaller primary tumour (57 (46%) screened v 11 (10%) controls had tumours less than or equal to 11 mm in diameter, p less than 0.0001), and less often had axillary nodal metastases (104 (83%) screened v 71 (57%) controls node negative, p less than 0.0001). After adjustment for the smaller size of the primary tumour compared with control cancers, those cancers detected by screening were less likely to have axillary nodal metastases (odds ratio 0.44, 95% confidence interval 0.23 to 0.84), poor histological differentiation (0.20, 0.08 to 0.49), high mitotic counts (0.38, 0.15 to 0.97), tumour necrosis (0.45, 0.22 to 0.93) or to be of the ductal histological type (0.46, 0.22 to 0.95). They had low oestrogen receptor (0.29, 0.12 to 0.70) and progesterone receptor (0.35, 0.17 to 0.92) concentrations less often and had smaller S phase fractions (0.72, 0.55 to 0.96) than control cancers. CONCLUSIONS--Even after adjustment for the smaller size of screen detected breast cancers, their histological and cytometric features suggest low malignant potential. They may also be less likely to metastasise to axillary lymph nodes than cancers found outside screening.     

Plasmid profiles of Lactobacillus plantarum spp. found as contaminants in Hemerocallis plant tissue cultures

Plasmid profiles of 30 strains of Lactobacillus plantarum isolated from 3-year-old tissue cultures and surface sterilized stem sections of glasshouse grown plants of three different varieties of Hemerocallis were compared to pinpoint the source of Lact. plantarum contamination in plant tissue cultures. Since the profiles of strains isolated from in vitro and in vivo plant material were generally identical, it is thought that the source of Lact. plantarum contamination is the plant material used to initiate plant tissue cultures.