Mammary-derived Growth Inhibitor Research Articles

P-156 - Targeting mammary-derived growth inhibitor for positron emission tomography imaging of breast cancer brain metastasis

P-156 - Targeting mammary-derived growth inhibitor for positron emission tomography imaging of breast cancer brain metastasis

Identification of novel polymorphism in mammary-derived growth inhibitor gene of water buffalo and its expression analysis in the mammary gland

Mammary-derived growth inhibitor (MDGI), a member of the lipophilic family of fatty acid-binding proteins, plays an important role in the development, regulation, and differentiation of the mammary gland. The aim of the study was to identify polymorphism in the MDGI gene and its expression analysis in the mammary gland at various stages of lactation, in Indian buffalo. Nucleotide sequence analysis of MDGI gene in different breeds of riverine and swamp buffaloes revealed a total of 16 polymorphic sites and one Indel. Different transcription factor binding sites were predicted for buffalo MDGI gene promoter sequence, using online tools and in-silico analysis indicating that the SNPs in this region can impact the gene expression regulation. Phylogenetic analysis exhibited the MDGI of buffalo being closer to other ruminants like cattle, yak, sheep, and goats. Further, the expression analysis revealed that buffalo MDGI being highly expressed in well-developed mammary glands of lactating buffalo as compared to involution/non-lactating and before functional development to start the milk production stage in heifers. Stage-specific variation in expression levels signifies the important functional role of the MDGI gene in mammary gland development and milk production in buffalo, an important dairy species in Southeast Asia.

Tumor-Targeting Peptides: The Functional Screen of Glioblastoma Homing Peptides to the Target Protein FABP3 (MDGI).

We recently identified the glioblastoma homing peptide CooP (CGLSGLGVA) using in vivo phage display screen. The mammary-derived growth inhibitor (MDGI/FABP3) was identified as its interacting partner. Here, we present an alanine scan of A-CooP to investigate the contribution of each amino acid residue to the binding to FABP3 by microscale thermophoresis (MST) and surface plasmon resonance (SPR). We also tested the binding affinity of the A-CooP-K, KA-CooP, and retro-inverso A-CooP analogues to the recombinant FABP3. According to the MST analysis, A-CooP showed micromolar (KD = 2.18 µM) affinity to FABP3. Alanine replacement of most of the amino acids did not affect peptide affinity to FABP3. The A-CooP-K variant showed superior binding affinity, while A-[Ala5]CooP and A-[Ala7]CooP, both replacing a glycine residue with alanine, showed negligible binding to FABP3. These results were corroborated in vitro and in vivo using glioblastoma models. Both A-CooP-K and A-CooP showed excellent binding in vitro and homing in vivo, while A-[Ala5]CooP and control peptides failed to bind the cells or home to the intracranial glioblastoma xenografts. These results provide insight into the FABP3–A-CooP interaction that may be important for future applications of drug conjugate design and development.

Altered Expression of Three EGFR Posttranslational Regulators MDGI, MIG6, and EIG121 in Invasive Breast Carcinomas.

Epidermal growth factor receptor (EGFR) signalling is a highly regulated process with a tight balance between receptor activation and inactivation in invasive breast carcinomas (IBCs) particularly in triple-negative carcinomas (TNC). Clinical trials using anti-EGFR therapies are actually performed although no activating alterations (mutations, amplifications, or rearrangements) of EGFR have been clearly recognized in order to identify new targeted modalities for IBCs. We explored mammary-derived growth inhibitor (MDGI), estrogen-induced gene-121 (EIG121), and mitogen-induced gene-6 (MIG6), three posttranslational EGFR trafficking molecules implicated in EGFR spatiotemporal regulatory pathway. We quantified MDGI, EIG121, and MIG6 at mRNA levels by using real-time quantitative RT-PCR in a series of 440 IBCs and at protein levels by using immunohistochemistry in a series of 88 IBCs. Results obtained by RT-PCR showed that in IBCs, MDGI, MIG6, and EIG121 mRNA were mainly underexpressed (25.7%, 45.0%, and 16.1%, respectively) particularly in the TNC subtype for EIG121 (60.3%). We also observed mRNA overexpression of MDGI and EIG121, respectively, in 12.7% and 22.3% of IBCs. These altered mRNA expressions were confirmed at the protein level. Some links were found between expression patterns of these three genes and several classical pathological and clinical parameters. Only EIG121 was found to have a prognostic significance (p = 0.0038). Altered expression of these three major EGFR posttranslational negative regulators could create an aberrant EGFR-mediated oncogenic signalling pathway in IBCs. MDGI, MIG6, and EIG121 expression status also may be potential useful biomarkers (sensitivity or resistance) in targeted EGFR therapy.

PCR-SSCP analysis of MDGI gene and its association with milk production traits in river buffalo (Bubalus bubalis)

In this study, we investigated the genetic variation within 3′UTR of Mammary-Derived Growth Inhibitor (MDGI) gene of buffalo using PCR-SSCP and sequencing; and also analyzed association of polymorphism with the milk production traits. The study revealed two conformational patterns, ‘A’ and ‘B’ among 234 Mehsana buffaloes maintained with their records in the field and at farm. The frequency of SSCP variant ‘A’ was found to be invariably high in the buffalo population under study. Further, association analysis of SSCP variants with various milk production and milk quality traits indicated no significant effect on any of the traits investigated. Sequencing of SSCP variant ‘A’ showed homozygous G/G and A/A and ‘B’ had heterozygous G/C and A/G at positions +124 and +140 respectively, in the 3′UTR of buffalo MDGI. The preliminary results showed the substantial variations in the distribution of SSCP variants' frequencies within Mehsana buffaloes, however these variants had non-significant association with milk yield, fat yield and fat percentage in Mehsana buffaloes.

Mammary-Derived Growth Inhibitor Targeting Peptide-Modified PEG-PLA Nanoparticles for Enhanced Targeted Glioblastoma Therapy.

Targeting delivery of chemotherapeutics to neovasculature represents a promising means for tumor therapy since angiogenesis has been a featured hallmark of glioblastma. However, anti-angiogenic therapy would induce the occurrence of metastatic tumor and even neoplasm recurrence. Simultaneous targeting of tumor cells and neovasculature perfectly overcome such defects and has been proven to be an efficacious strategy for suppressing tumor growth. In the present study, a tumor homing peptide CooP selective binding to mammary-derived growth inhibitor that overexpressed in glioma cells and blood vessel endothelial cells was decorated on the surface of paclitaxel-loading PEG-PLA nanoparticles (NP-PTX) to obtain the dual targeting nanovector CooP-NP-PTX. In vitro antiproliferation study showed that HUVEC cells and U87MG cells were much more sensitive to CooP-NP-PTX than NP-PTX. In vivo imaging demonstrated that CooP-NP accumulated more selectively and penetrated deeper into the tumor site. In addition, the glioma-bearing mice treated with CooP-NP-PTX achieved the longest survival time compared to NP-PTX and Taxol. The findings observed above indicated that CooP peptide-functionalized anti-neoplastic agent-loaded nanoparticles might possess promising potential for glioblastoma therapy.

Highlights of This Issue

Due to the poor prognosis of glioblastoma patients, innovative treatment approaches are urgently needed. Here, Hyvönen and colleagues have identified a novel homing peptide that recognizes tumor vessels and invasive tumor satellites in gliomas. This peptide was not only shown to be effective in SPECT/CT-imaging but also in selective delivery of chemotherapeutics that prolonged the lifespan of invasive brain tumor–bearing mice. Mammary-derived growth inhibitor (MDGI/H-FABP/FABP3), the expression of which is positively correlated with the histological grade of human brain tumors, was found to be the interacting partner for this novel peptide in gliomas.The presence of cancer stem cells (CSC) contributes to the chemoresistance and relapse of basal-like breast cancers (BLBC). Niclosamide inhibits the Wnt/β-catenin pathway involved in CSC regulation. TRA-8 antibody, specific to TRAIL death receptor 5, is cytotoxic to BLBC cell lines and CSC. In this study, Londoño-Joshi, Arend and colleagues demonstrate that niclosamide in combination with TRA-8 reduced Wnt/β-catenin activity and produced increased cytotoxicity against BLBC cell lines, tumor xenografts, and patient-derived BLBC tumor cells. These results suggest that niclosamide or its analogs may be useful for the treatment of BLBC patients.Genetic alterations affecting the histone methyltransferase activity of EZH2 have been implicated as oncogenic drivers of a spectrum of human cancers; this includes heterozygous expression of point mutations, within EZH2, in a subset of non-Hodgkin lymphoma (NHL) patients. Knutson, Kawano and colleagues report detailed, preclinical characterization of a potent, selective, orally bioavailable EZH2 inhibitor, EPZ-6438 (also known as E7438), which is currently in phase I clinical testing. Twice daily oral administration of this compound to mice bearing human NHL xenografts, with different EZH2 mutations, resulted in profound and durable tumor regression, thus illustrating the potential clinical utility of this drug.The heterogeneous delivery of drugs in tumors has been a challenge for achieving effective treatment outcome. In the present study, Thurber and colleagues developed fluorescent PARP inhibitor olaparib drug conjugates that retain their target binding but are designed with different physiochemical and, thus, pharmacokinetic properties. The drug distribution in cell culture and tumor xenografts was followed with temporal resolution of seconds and subcellular spatial resolution. These measurements, including in vivo permeability of small molecule drugs, can be used directly in predictive pharmacokinetic models for the design of therapeutics and companion imaging agents.

Novel target for peptide-based imaging and treatment of brain tumors.

Malignant gliomas are associated with high mortality due to infiltrative growth, recurrence, and malignant progression. Even with the most efficient therapy combinations, median survival of the glioblastoma multiforme (grade 4) patients is less than 15 months. Therefore, new treatment approaches are urgently needed. We describe here identification of a novel homing peptide that recognizes tumor vessels and invasive tumor satellites in glioblastomas. We demonstrate successful brain tumor imaging using radiolabeled peptide in whole-body SPECT/CT imaging. Peptide-targeted delivery of chemotherapeutics prolonged the lifespan of mice bearing invasive brain tumors and significantly reduced the number of tumor satellites compared with the free drug. Moreover, we identified mammary-derived growth inhibitor (MDGI/H-FABP/FABP3) as the interacting partner for our peptide on brain tumor tissue. MDGI was expressed in human brain tumor specimens in a grade-dependent manner and its expression positively correlated with the histologic grade of the tumor, suggesting MDGI as a novel marker for malignant gliomas.

Tumour homing peptide-functionalized porous silicon nanovectors for cancer therapy

Tumour targeting nanoparticles (NPs) have demonstrated great potential for enhancing anticancer drug delivery to tumour sites and for reducing the side effects of chemotherapy. However, many nanoparticulate delivery systems still lack efficient tumour accumulation. In this work, we present a porous silicon (PSi) nanovector functionalized with a tumour-homing peptide, which targets the mammary-derived growth inhibitor (MDGI) expressing cancer cells both in vitro and in vivo, thereby enhancing the accumulation of the NPs in the tumours. We demonstrated that the tumour homing peptide (herein designated as CooP) functionalized thermally hydrocarbonized PSi (THCPSi) NPs homed specifically to the subcutaneous MDGI-expressing xenograft tumours. The THCPSi-CooP NPs were stable in human plasma and their uptake by MDGI-expressing cancer cells measured by confocal microscopy and flow cytometry was significantly increased compared to the non-functionalized THCPSi NPs. After intravenous injections into nude mice bearing MDGI-expressing tumours, effective targeting was detected and THCPSi-CooP NPs showed ∼9-fold higher accumulation in the tumour site compared to the control THCPSi NPs. Accumulation of both NPs in the vital organs was negligible.

83 EFFECTS OF HUMAN INTERFERON-α ON GENE EXPRESSION IN THE BOVINE ENDOMETRIUM IN COMPARISON TO DAYS 15 AND 18 OF PREGNANCY

Interferon-τ (IFNT), a Type-I interferon (IFN), is the pregnancy recognition signal produced by the ruminant conceptus (Godkin et al. 1984; Hansen et al. 1985; Helmer et al. 1987; Spencer et al. 2007). In addition to these specific functions of IFNT in ruminants, many studies suggest that IFNs play a general role in establishment of pregnancy and conceptus attachment/implantation in most mammalian species (Bazer et al. 2009; Bazer et al. 2010; Johnson et al. 2009; Roberts et al. 2008). To characterise the effects of prototype Type-I IFNs on bovine endometrium, in experiment one, Simmental heifers were treated from Day 14 to Day 16 of the oestrous cycle with a rod-shaped intrauterine device releasing human interferon-α (IFNA) or placebo lipid extrudates or PBS only as controls (n = 4 each). Lipid formulation and concentration of human IFNA were adjusted to release 8–9 × 107 IU of IFNA over a period of 2 days in in vitro release experiments. On Day 16, endometrial biopsy samples were collected after flushing the uterus. In experiment 2, endometrial tissue samples were obtained on Day 12, 15 and 18 post-mating from nonpregnant or pregnant heifers. All samples from both experiments were analysed with an Affymetrix Bovine Genome Array (Santa Clara, CA). In experiment one, IFNA treatment resulted in differential gene expression in the bovine endometrium. Significant differences were found between the IFNA group and both control groups, whereas no differences were observed between the placebo and the PBS control group. In experiment 2, differentially expressed genes were found between pregnant and nonpregnant endometria on Day 15 and 18, but not on Day 12, with many of them known IFN-stimulated genes. The comparison of the data sets from both experiments showed very similar gene expression changes for most of the typical IFN-stimulated genes. In addition, several genes were identified which were differentially expressed after IFNA treatment but not different at Day 15 or 18 of pregnancy compared with nonpregnant animals. Conversely, some genes were found as differentially expressed during pregnancy but not after IFNA treatment. Differential expression of selected genes was verified by quantitative real-time PCR and 4 genes, namely jumonji C domain containing histone demethylase 1 homologue D (JHDM1D), indoleamine 2,3-dioxygenase 1 (IDO1), fatty acid binding protein 3, muscle and heart (mammary-derived growth inhibitor) (FABP3) and dickkopf homologue 1 (DKK1), were selected for localization of mRNA expression in endometrial tissue sections. The findings of this study suggest that there may be differential effects of bovine IFNT compared with human IFNA and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT. This study was supported by the German Ministry for Education and Research (BMBF, FUGATO-plus, COMPENDIUM) and the German Research Foundation (DFG FOR478). The authors are part of the European Union COST action GEMINI.

Mammary-derived growth inhibitor (MDGI) interacts with integrin α-subunits and suppresses integrin activity and invasion

The majority of mortality associated with cancer is due to formation of metastases from the primary tumor. Adhesion mediated by different integrin heterodimers has an important role during cell migration and invasion. Protein interactions with the β1-integrin cytoplasmic tail are known to influence integrin affinity for extracellular ligands, but regulating binding partners for the α-subunit cytoplasmic tails have remained elusive. In this study, we show that mammary-derived growth inhibitor (MDGI) (also known as FABP-3 or H-FABP) binds directly to the cytoplasmic tail of integrin α-subunits and its expression inhibits integrin activity. In breast cancer cell lines, MDGI expression correlates with suppression of the active conformation of integrins. This results in reduced integrin adhesion to type I collagen and fibronectin and inhibition of cell migration and invasion. In tissue microarray of 1331 breast cancer patients, patients with MDGI-positive tumors had more favorable 10-year distant disease-free survival compared with patients with MDGI-negative tumors. Our data indicate that MDGI is a novel interacting partner for integrin α-subunits, and its expression modulates integrin activity and suppresses cell invasion in breast cancer patients. Retained MDGI expression is associated with favorable prognosis.

Abstract 56: Differential expression of fatty-acid-binding proteins 3 and 5 among African American and Caucasian women with breast cancer

Abstract Breast cancer is a major cause of mortality among middle-aged and elderly women. It is well documented that African-American (AA) patients tend to present with more advanced states of breast cancer and have lower survival rates than Caucasian-Americans (CA). Additionally, the age specific breast cancer incidence rate for AA women under age 35 is greater than twice the rate for CA of similar age, and the mortality rate is more than three times greater. Some of this disparity is due to socioeconomic factors; however, biological factors may also be involved. Altered expression of intracellular fatty-acid binding proteins (FABPs) has been implicated in the progression of breast cancer. FABP3 (heart/muscle type), also known as mammary-derived growth inhibitor (MDGI), is associated with differentiation of breast tissue, and is downregulated in breast cancer tissues. FABP5 (epidermal type) is upregulated in breast cancer tissues as a possible compensation for FABP3's decrease. Therefore, we hypothesized that AA malignant breast tissue samples would contain lower levels of FABP3, and higher levels of compensatory FABP5. We determined levels of the aforementioned proteins and the corresponding mRNA in paired breast tissue samples from AA and CA women by Western blot analysis and RT-PCR. Data was divided into three groups- ages 17-45, ages 45-65, and ages 66 and up. In ages 17-45, FABP3 protein levels were higher in AA malignant tissues relative to CA malignant, and FABP5 protein levels were similar between racial groups. Also, FABP3 levels were decreased in AA malignant tissues compared to AA normal. In ages 46-65, levels of FABP3 protein, and of FABP5 protein and mRNA, were higher in AA malignant tissues relative to CA malignant. Furthermore, FABP3 protein levels were increased in AA malignant tissues relative to AA normal tissues. In ages 66 and up, levels of both FABPs were similar between AA malignant and CA malignant tissues. However, mRNA levels of both FABPs were significantly increased in the CA malignant tissues compared to AA malignant. In addition, levels of FABP3 and FABP5 were increased in the AA malignant tissues relative to the AA normal tissues. Thus, it appears that the conventional knowledge regarding FABP3 downregulation in breast cancer may only apply to AA cancer patients below age 45. Furthermore, in ages above 66, AA breast cancer patients are at a significant disadvantage compared to their CA counterparts, as it relates to expression of FABPs 3 and 5. Further study is needed to elucidate the mechanisms involved in this differential FABP expression pattern between the racial groups, and its implication on the survival disparity. 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 56.

Mammary-Derived Growth Inhibitor Alters Traffic of EGFR and Induces a Novel Form of Cetuximab Resistance

Only few predictive factors for the clinical activity of anti-epidermal growth factor receptor (EGFR) therapy are available. Mammary-derived growth inhibitor (MDGI) is a small cytosolic protein suggested to play a role in the differentiation of epithelial cells. Here, we have investigated the effect of MDGI expression on the EGFR signaling and cetuximab responsiveness of cancer cells. MDGI mRNA expression was investigated in clinical breast and lung cancer samples and in nontransformed and malignant cell lines. The effect of ectopic expression of MDGI on EGFR, ErbB2, and integrin function and traffic was investigated in breast and lung cancer cell lines using multiple methods. The effect of anti-EGFR agents on these cells were tested by cell proliferation measurements and by assessing tumor growth of breast cancer cells in cetuximab treated and control athymic nude mice. Here, we show that although MDGI is absent in cultured cell lines because of epigenetic silencing, MDGI mRNA is expressed in 40% of clinical breast carcinomas and 85% of lung cancers. Ectopic expression of MDGI rendered breast and lung cancer cells resistant to the anti-EGFR antibody cetuximab in vitro and in an orthotopic breast cancer xenograft model in vivo. When expressed in cancer cells, MDGI induces intracellular accumulation of EGFR, but not ErbB2, and the internalized receptor is phosphorylated and not degraded. MDGI-driven inherent desensitization of cancer cells is a novel molecular mechanism for resistance to the anti-EGFR antibody therapy, and MDGI may be a biomarker for responsiveness to anti-EGFR antibody therapy.

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Expression of mammary derived growth inhibitor (MDGI) results in phenotypic reversal in breast cancer

Expression of mammary derived growth inhibitor (MDGI) results in phenotypic reversal in breast cancer

Construction and Evaluation of Directionally Cloned cDNA Libraries from Lactating and Non-lactating Mammary Gland of River Buffalo (Bubalus bubalis): A Resource for Gene Identification in Bubaline Genome

Mukesh, M., Kataria, R.S., Kumar, V., Pandey, D., Sodhi, M., Ahlawat, S.P.S., Sobti, R.C. and Mishra, B.P. 2008. Construction and evaluation of directionally cloned cDNA libraries from lactating and non-lactating mammary gland of river buffalo (Bubalus bubalis): A resource for gene identification in bubaline genome. J. Appl. Anim. Res., 33: 81–84. Directionally cloned cDNA libraries from lactating (Lac-MG) and non-lactating (NLac-MG) buffalo mammary gland were constructed and characterized. The total number of independent primary clones were: 8.0 × 104 and 1.1 × 105 for Lac-MG and Nlac-MG cDNA libraries, respectively, with >90% of the clones having cDNA inserts. The occurrence of large cDNA insert length (average 0.90 Kb: range 0.45–2.5 Kb in Lac-MG and average 0.57 Kb: range 0.32–1.8 Kb in NLac-MG cDNA libraries) was indicative of the good quality status of both the libraries. The single-pass sequencing of randomly selected cDNA clones found several full-length cDNA sequences with complete 5′ and 3′ UTR. The initial sequence analysis showed several clones harboring milk protein, ribosomal and regulatory gene transcripts like ubiquitin, mammary derived growth inhibitor, lipoprotein lipase etc. The two cDNA libraries can be utilized as gene resources for cataloguing and annotation of mammary gland transcripts/ESTs in river buffaloes.

Activation of Stat5 and induction of a pregnancy‐like mammary gland differentiation by eicosapentaenoic and docosapentaenoic omega‐3 fatty acids

The protective effect of early pregnancy against breast cancer can be attributed to the transition from undifferentiated cells in the nulliparous to the differentiated mature cells during pregnancy. Considerable evidence suggests strongly that the n-3 polyunsaturated fatty acid (PUFA) content of adipose breast tissue is inversely associated with an increased risk of breast cancer. Here, we report that there was a decrease in the n-6/n-3 PUFA ratio and a significant increase in concentration of n-3 PUFA docosapentaenoic acid and eicosapentaenoic acid in the pregnant gland. The functional role of n-3 PUFAs on differentiation was supported by the studies in the fat-1 transgenic mouse, which converts endogenous n-6 to n-3 PUFAs. Alternation of the n-6/n-3 ratio in favor of n-3 PUFA, and particularly docosapentaenoic acid, in the mammary gland of fat-1 mouse resulted in development of lobulo-alveolar-like structure and milk protein beta-casein expression, mimicking the differentiated state of the pregnant gland. Docosapentaenoic acid and eicosapentaenoic acid activated the Jak2/Stat5 signaling pathway and induced a functional differentiation with production of beta-casein. Expression of brain type fatty acid binding protein brain type fatty acid binding protein in virgin transgenic mice also resulted in a reduced ratio of n-6/n-3 PUFA, a robust increase in docosapentaenoic acid accumulation, and mammary differentiation. These data indicate a role of mammary derived growth inhibitor related gene for preferential accumulation of n-3 docosapentaenoic acid and eicosapentaenoic acid in the differentiated gland during pregnancy. Thus, alternation of n-6/n-3 fatty acid compositional ratio in favor of n-3 PUFA, and particularly docosapentaenoic acid and eicosapentaenoic acid, is one of the underlying mechanisms of pregnancy-induced mammary differentiation.

Isolation and Sequence Characterization of Mammary Derived Growth Inhibitor Gene of Riverine Buffalo (Bubalus Bubalis)

In this study, attempts have been made to identify and characterize water buffalo (Bubalus bubalis) mammary derived growth inhibitor (MDGI) gene, isolated from a mammary gland cDNA library of lactating buffalo. The complete MDGI cDNA was of 698 nucleotides, consisting 61 nucleotides in 5′ UTR, coding region of 402 nucleotides, and 235 nucleotides representing the 3′ UTR. Comparison of nucleotide and deduced amino acid sequence data with that of MDGI//fatty acid binding protein (FABP) of other species shows three buffalo specific nucleotide changes while seven nucleotide changes were common to cattle and buffalo. Buffalo and cattle MDGI had 100% amino acid sequence similarity, which also shared three amino acid changes: 34 (Ala-Gly), 109 (Leu-Met), and 132 (Glu-Gln) as compared to other species. Comparison with FABPs reported from other cattle tissues revealed highest amino acid sequence similarity with FABP-heart (100%) and least with FABP-liver (20.5%). Phylogenetic analysis revealed cattle MDGI to be closest to buffalo, while mouse MDGI was distantly placed, whereas different tissue derived FABPs of cattle showed FABP-heart closest and FABP-epidermis most distantly placed from buffalo MDGI. This report also differs from the earlier findings that MDGI is intermediate of FABP-heart and adipose.

Bone Marrow Stromal Cell-derived Growth Inhibitor Inhibits Growth and Migration of Breast Cancer Cells via Induction of Cell Cycle Arrest and Apoptosis

Genes encoding growth-inhibitory proteins are postulated to be candidate tumor suppressors. The identification of such proteins may benefit the early diagnosis and therapy of tumors. Here we report the cloning and functional characterization of a novel human bone marrow stromal cell (BMSC)-derived growth inhibitor (BDGI) by large scale random sequencing of a human BMSC cDNA library. Human BDGI cDNA encodes a 477-amino acid residue protein that shares high homology with rat and mouse pregnancy-induced growth inhibitors. The C-terminal of BDGI is identical to a novel human pregnancy-induced growth inhibitor, OKL38. BDGI is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved family of BDGI-like proteins. BDGI overexpression inhibits the proliferation, decreases anchorage-dependent growth, and reduces migration of MCF-7 human breast cancer cells, whereas down-regulation of BDGI expression promotes the proliferation of MCF-7 and HeLa cervix epitheloid carcinoma cells. Interestingly, the inhibitory effect of BDGI on MCF-7 cells is more potent than that of OKL38. We demonstrate that BDGI induces cell cycle arrest in S phase and subsequent apoptosis of MCF-7 cells, which is likely to account for the antiproliferative effects of BDGI. This process may involve up-regulation of p27Kip1 and down-regulation of cyclin A, Bcl-2, and Bcl-xL. The inhibitory effect of BDGI on cell proliferation and the induction of apoptosis were also observed in A549 lung cancer cells but not HeLa cells. These results indicate that BDGI might be a growth inhibitor for human tumor cells, especially breast cancer cells, possibly contributing to the development of new therapeutic strategies for breast cancer.

Heart-type fatty acid binding proteins are upregulated during terminal differentiation of mouse cardiomyocytes, as revealed by proteomic analysis.

At birth, the cardiomyocytes in the mouse neonatal heart still retain their ability to proliferate. However, this lasts only a few days and then the cardiomyocytes irreversibly lose their potential to divide. It is still not fully understood what factors are involved in the cessation of cardiomyocyte proliferation. Using proliferating cell nuclear antigen (PCNA) antibodies, we established that cardiomyocytes could divide extensively in 2-day-old mouse neonatal hearts and to a lesser extent in 6-day-old hearts. By 13 days, the cardiomyocytes have mostly stopped dividing. Comparative two-dimensional gel electrophoresis (2-DE) was performed on total proteins extracted from the 2-day- and 13-day-old hearts, in order to identify peptides that might be involved in the inhibition of cardiomyocyte proliferation. Using matrix-assisted laser desorption ionization mass spectroscopy (MALDI-TOF), we identified two protein spots that have the same molecular weight (approximately 14 kDa) but different pIs (5.9 and 6.1). Mass spectra analysis determined the proteins to be isoforms of the heart-type fatty acid binding protein (H-FABP). The pI 6.1 H-FABP is also known as mammary-derived growth inhibitor (MDGI; Specht et al. 1996). MGDI is a breast tumour growth suppressor gene capable of inhibiting tumour cell proliferation (Huynh et al. 1995). Both H-FABP isoforms were expressed in 2-day-old hearts but became strongly upregulated in 13-day-old hearts. We examined whether H-FABPs and PCNA were coexpressed in 2-, 6- and 13-day-old heart histological sections, using MDGI antibodies. The antibody could detect both forms of H-FABPs. It was established that there was a correlation between an increase in H-FABP expression and a decrease in PCNA expression. Hence, we tentatively propose that H-FABP isoforms are involved in regulating cardiomyocyte growth and differentiation in mouse neonatal hearts.