Mitochondrial lineages reveal intense gene flow between Iberian wild boars and South Iberian pig breeds.

The phylogeography of wild boars (WB) and domestic pigs (Sus scrofa) has contributed important insights into where and when domestication occurred. The geographic distribution of two core haplotypes (E1a and E1c) of the main European phylogenetic clade suggests that Central Europe was an early domestication centre, although the complexity of the pattern does not exclude the possibility that multiple domestication events occurred in different regions. To investigate the relationships among WB and domestic pig breeds in Iberia, a fragment of the mitochondrial DNA control region from a large sample (n=409) of WB and local pig breeds was co-analysed with published sequences from other European populations. The Iberian sample revealed a high frequency of a sub-cluster (E1c) of the European haplogroup E1 in 77% of total Iberian samples, 96% of WB, 90% of Alentejano (Portugal) and 87% of Iberian breed pigs (Spain; Black Hairy, Black Hairless and Red varieties). Low genetic distance (F'(ST) = 0.105) was observed between Alentejano (Portugal) and Iberian breed pigs (Spain). Alentejano and Iberian breed pigs showed low genetic distances to both Iberian and Central European WB (average F'(ST) =0.345 and 0.215, respectively). This pattern suggests that early pig husbandry in the Iberian Peninsula did not solely rely on imported Central European stock, but also included the recruitment of local WB.

The mtDNA catalogue of all Portuguese autochthonous goat (Capra hircus) breeds: high diversity of female lineages at the western fringe of European distribution.

Goat is believed to be the first true livestock domesticated and, apart from its historical importance, keeps playing an essential economic role in very diverse human societies. We have analysed the female gene pool of all Portuguese autochthonous breeds (Bravia, Serrana, Charnequeira, Serpentina and Algarvia) through the mtDNA HVI sequencing of 288 unrelated animals sampled throughout the country. All breeds proved to be extremely diverse (average haplotype diversity of 0.977), in contrast with the Portuguese peripheral geographic situation in the distribution range of the species. Moreover, observed genetic distances between breeds do not correlate with microgeography inside Portugal. These observations are consistent with recurrent refreshment of the breeding stock through the introduction of exotic animals. Fitting the new data into the still loosely defined female genetic pool landscape of goats, all Portuguese animals, one sample excepted (belonging to Bravia), are classified into haplogroup A and haplotype sharing is geographically very sparse, including a Far East match. Our results confirm that goats stand out among most of domesticates as exceptionally diverse and showing an unparalleled degree of mobility of animals (at least females) used for reproductive purposes.

Cloning and characterization of ectopically expressed transcripts for the actin-binding protein MIPP in mouse mammary carcinomas.

Mipp is a kelch-related, placental-specific gene that is ectopically expressed in many BALB/c mouse mammary carcinomas of various etiologies. The Kelch family encompasses proteins that are emerging as key links between microfilaments and a variety of cellular structures and functions. Mouse mammary tumors express two mipp transcripts (2.2 and 5.6 kb). We cloned the 2.2 kb mipp mRNA and analysed the product of its 1.7 kb ORF. The 584 residue MIPP protein has an N-terminal BTB domain and six C-terminal tandem Kelch repeats. Despite expression of two mipp RNAs, only a single MIPP protein is expressed in mammary tumors. MIPP protein binds to microfilaments in vitro and co-immunoprecipitates with actin. MIPP co-localized with concanavalin A at the endoplasmic reticulum, suggesting that MIPP might mediate interactions between microtubules and actin filaments. Because MIPP expression is widespread in mouse mammary tumors, it might contribute to tumorigenesis. Although MIPP had little effect on the growth rate of human breast cell lines following transfection, it greatly reduced the formation of duct-like structures on reconstituted basement membrane. Our results suggest that MIPP could contribute to malignant progression in the mouse mammary epithelial cells by perverting their response to cues from the extracellular matrix.

Genes, chromatin, and breast cancer: an epigenetic tale.

The production of heritable changes in gene expression is the driving force in the development and progression of breast cancer. Such changes can result from mutations or from epigenetic events such as hypermethylation of DNA and hypoacetylation of histones. Histone acetylation and DNA methylation are major determinants of chromatin structure, and chromatin structure is a primary regulator of gene transcription. Cancer cells frequently contain both mutated genes and genes with altered expression due to one or more epigenetic mechanisms. This review describes the epigenetic changes that disrupt normal chromatin architecture and modify the expression of key genes in breast cancer cells. The structural integrity of the latter genes is usually intact, but their expression has been substantially altered due to methylation in their promoter region or deacetylation of histones that interact with their promoter region or both mechanisms. Genes affected by epigenetic changes in breast cancers include HoxA5, p21WAF, gelsolin, BRCA1, BRCA2, E-cadherin, steroid hormone receptors, and retinoic acid receptor II. Because these epigenetic modifications are usually reversible by treatment with certain drugs, they represent vulnerabilities in the cancer cell that can be exploited as novel targets for new prevention and therapeutic strategies.

Downregulation of gelsolin correlates with the progression to breast carcinoma.

The actin cytoskeleton underlies several normal cellular functions and is deranged during carcinogenesis. Gelsolin, a multifunctional actin-binding protein, is downregulated in several types of tumors and its abnormal expression is one of the most common defects noted in invasive breast carcinoma (ICA). This study utilizes immunohistochemistry to examine the expression of gelsolin in 95 ICA, 59 ductal carcinoma in situ (DCIS) and 36 benign lesions, including 17 atypical ductal hyperplasia (ADH). Cytoplasmic staining was scored as positive, reduced or negative. Gelsolin expression was then correlated with patient's age, tumor size, histologic grade and lymph node status. All unremarkable breast biopsies, 88% of ADH, 44% of DCIS and 28% of ICA were positive for gelsolin. This represents a significant difference among the groups (p = < 0.0001) and the trend towards reduced gelsolin with the progression to ICA is significantly linear (p = < 0.0001). For invasive carcinoma, patients older than 44 years were significantly more likely to have decreased expression of gelsolin than patients 44 years old and younger (p = 0.007). Bivariate analysis showed no correlation of gelsolin expression with lymph node status (p = 0.62), tumor size (p = 0.10), histologic grade (p = 0.42), estrogen receptor status (p = 1.0) or other clinicopathologic parameters. In clinical follow-up, there were 18 breast tumor related deaths within a median follow-up time of 4.2 years. Survival analysis indicated that the level of gelsolin expression may be associated with survival (p = 0.06). In summary, the frequency of gelsolin deficiency increases significantly with progression from ADH to DCIS to ICA. Additionally, gelsolin expression may be an independent marker of prognosis.

The mouse mammary gland requires the actin-binding protein gelsolin for proper ductal morphogenesis.

Gelsolin is an actin-binding/severing protein expressed in intracellular and secreted forms. It is a major regulator of the form and function of the actin cytoskeleton in most all cells. Here we demonstrate that female mice with a targeted deletion of the gelsolin gene (Gsn-/-) have defects in mammary gland morphogenesis. Two distinct defects were identified in the gelsolin-null mammary gland. First, the mammary anlage from Gsn-/- mice failed to elongate at the onset of puberty and remained rudimentary until approximately 9 weeks of age, early block (Gsn-/-(EB)). Second, after the mammary epithelium had filled the mammary fat pad, a complete lack of terminal branching, or late block, was observed (Gsn-/-(LB)). The Gsn-/-(EB) was seen in 70% of Gsn-/- mice and appeared to be dependent on a modifier gene(s) in addition to the loss of gelsolin. Gsn-/-(LB) was observed in all Gsn-/- mice. Terminal end buds (TEBs) were not evident in the mammary anlage from Gsn-/-(EB) mice until approximately 9 weeks of age. Cellular proliferation in the terminal ductal regions of Gsn-/-(EB) females was detected by bromodeoxyuridine incorporation, but was less than that found in the TEBs of age-matched controls. In mice deficient for gelsolin, mammary gland architecture was unaltered at the histological level. Lobuloalveolar development was delayed in response to pregnancy in mammary glands of Gsn-/- mice but was otherwise normal. Lactation and involution in the gelsolin-null animals were similar to those of wild-type mice. Transplantation of epithelium devoid of gelsolin into a wild-type (GsnWT) mammary fat pad resulted in proper arborization of the ductal tree. Transplantation of GsnWT epithelium into the Gsn-/- fat pad recapitulated the lack of terminal branching seen in Gsn-/- females. These results indicate that gelsolin is required in the mammary stroma for proper ductal morphogenesis. Our results provide the first evidence of an actin regulatory protein affecting mammary ductal growth through stromal-epithelial communication.

Down-regulation of gelsolin expression in human breast ductal carcinoma in situ with and without invasion.

Expression of gelsolin, an actin filament regulatory protein, in human breast ductal carcinoma in situ (DCIS) was analyzed by immunohistochemistry using a monoclonal antibody. Formalin-fixed paraffin-embedded tissues from 59 pure DCIS specimens and 33 DCIS specimens with associated invasive components were evaluated for gelsolin reactivity and compared to eight normal breast cases and 76 invasive breast cancers. The proportion of cases exhibiting negative/low expression of gelsolin in the epithelium was as follows -- normal, 0%; pure DCIS, 56%; DCIS associated with invasion, 58% in the DCIS component and 66% in the invasive component; invasive carcinoma, 70%. These data demonstrate that down-regulation of gelsolin expression in breast epithelium frequently parallels progression to malignancy. Testing gelsolin expression (normal vs. negative/low levels) in the DCIS lesions for associations with patient age or any of the following histopathologic parameters revealed no significant (95% probability level) correlations -- tumor size; pathologic (Van Nuys system) grade; nuclear grade; necrosis; presence of histologic calcifications; presence or type of adjacent benign lesions; architectural histologic pattern; and mammographic extent. Gelsolin loss was more commonly associated with mammographic soft tissue lesions as compared to calcified lesions (P = 0.009). A positive trend of borderline significance (P = 0.06) found in the DCIS with invasion group was a correlation between down-regulated gelsolin expression in the DCIS component and size (< versus > or = 15 mm) of the invasive tumor. In conclusion, reduced gelsolin protein is detectable in at least half of breast lesions which have progressed to DCIS. The trend between increasing gelsolin loss and malignant progression from normal epithelium to DCIS to invasive breast cancer (P < 0.0001) suggests additional investigation is needed to determine the potential of altered gelsolin expression as a marker for prognosis and for therapeutic interventions in breast cancer.

Concurrent deregulation of gelsolin and cyclin D1 in the majority of human and rodent breast cancers.

Decreased gelsolin and increased cyclin D1 are among the most common defects found in human and rodent breast cancers. Our purpose was to determine the frequency of concurrence of these 2 alterations in this malignancy. Our results demonstrate that gelsolin protein and mRNA were significantly reduced in 80-100% of rodent mammary carcinomas that developed spontaneously, following oncogene introduction, or after treatment with viral, chemical or hormonal agents. The reduction in gelsolin most likely occurs during the transition from preneoplasia to carcinoma because hyperplasias had normal levels of gelsolin whereas microtumors had reduced expression. Southern analysis revealed no major mutations in the gelsolin gene of tumors with low expression. Cyclin D1 mRNA was increased in 50-100% of these rodent mammary tumors, although the cyclin D1 gene was not amplified. By nuclear runon assay, downregulation of gelsolin in both human and mouse mammary cancer cells involved diminished transcription and, conversely, human breast cancer cells expressing high levels of cyclin D1 had increased initiation of cyclin D1 transcription compared with cyclin D1 low expressors. Thus, alteration in the rate of transcription appears to be an important factor underlying the dysfunction of these genes. According to our data, concurrent deregulation of gelsolin and cyclin D1 is highly prevalent among breast cancers of humans and rodents, with both defects present in 89% of the neoplasms analyzed in this study. In fact, most tumors in every rodent model of mammary tumorigenesis examined had the 2 alterations.

Epigenetic regulation of gelsolin expression in human breast cancer cells.

Gelsolin is a multifunctional, actin-binding protein that is greatly decreased in many transformed cell lines and tumor tissues, including breast cancers. Downregulation of gelsolin RNA occurs in most breast cancers of rats, mice, and humans, but gross mutations of the gelsolin gene have not been found. Here we demonstrate by PCR and RT-PCR analysis that there are no point mutations in putative regulatory regions or the entire coding region of the cytoplasmic isoform of the gelsolin gene in human breast cancer cells (BCC). To determine if epigenetic modification is involved in downregulating gelsolin expression in MDA-MB-231 (MDA231), MCF7, and T47D BCC, we have used Southern blot analysis, 5-azacytidine (5aza) treatment, and trichostatin A (TSA) treatment. Southern blot analysis performed on genomic DNA demonstrated altered CpG methylation within intron 1 in DNA from all BCC compared to normal, mortal human mammary epithelial cells (HMEC). Treatment of the BCC with 5aza converted the DNA restriction pattern to that seen in untreated HMEC genomic DNA and caused modest increases in gelsolin RNA and protein. Incubation with TSA, an inhibitor of histone deacetylase, induced a dramatic upregulation of gelsolin RNA and protein levels which preceded apoptotic death of all BCC within 48-60 h. Our data support a role for epigenetic changes in chromatin structure leading to downregulation of gelsolin expression in human breast cancer. To our knowledge, this is the first example of a tumor suppressor gene downregulated in human breast cancer by changes in histone acetylation.

Downregulated gelsolin expression in hyperplastic and neoplastic lesions of the prostate.

BACKGROUND: Because of its role in cell motility and growth regulation, gelsolin, an actin-binding protein, has been considered a tumor suppressor and a potential prognostic marker in some neoplasias, such as breast and bladder cancer. Little is known about its immunoexpression in prostatic adenocarcinoma (PCA). METHODS: Formalin-fixed, paraffin-embedded tissues of 72 prostatectomy specimens with adenocarcinoma and 8 nonneoplastic prostates from autopsies were stained with a gelsolin monoclonal antibody using the Avidin-biotin-peroxidase complex (ABC) method after microwave antigen retrieval. Immunoreactivity was evaluated in PCA, prostatic intraepithelial neoplasia (PIN), benign prostatic hyperplasia (BPH), and nonproliferative glandular tissue and stroma. The results were statistically analyzed. RESULTS: Consistent gelsolin immunoreactivity was seen in prostatic stromal cells, smooth muscle, endothelia, and nerves. Variable gelsolin expression was seen in 20-100% (average (A) = 65.5%) of glandular cells in nonproliferative tissue (N = 75); 0-50% (A = 9.7%) in BPH (N = 59); 0-80% (A = 8.9%) in PIN (N = 61); and 0-90% (A = 9.3%) in PCA (N = 71). The level of gelsolin expression in nonproliferative prostatic tissue was similar between prostates with PCA (A = 63.4%) and nonneoplastic prostates (A = 67.5%). The level of gelsolin expression did not correlate with age, tumor size, Gleason score, or stage. CONCLUSIONS: Gelsolin is decreased in PCA, PIN, and BPH in comparison to nonproliferative tissue. The role of this downregulation in the development of PCA is not clear. The similar reduction seen in PIN and BPH suggests that this event takes place indiscriminately in hyperplasia and early tumorigenesis in the prostate, which might limit its prognostic significance in PCA.

Widespread loss of gelsolin in breast cancers of humans, mice, and rats.

Down-regulation of gelsolin, an actin-binding protein, is frequently found in several types of transformed cells and tumors. The present study demonstrates that gelsolin protein and RNA were absent or markedly reduced in human breast cancer cell lines relative to "normal" mortal human mammary epithelial cells and benign, immortalized cell lines. Moreover, actin filaments were usually attenuated coincident with the reduction in gelsolin. Gelsolin was also missing or greatly decreased in 70% of 30 human sporadic, invasive breast carcinomas examined by immunocytochemistry and in 100% of virally induced mouse and chemically induced rat mammary carcinomas evaluated by Northern analysis. Southern analysis revealed no major mutations in the gelsolin gene of human breast cancer cells. Our results show that partial or total loss of gelsolin expression is common to the majority of breast cancers of diverse etiologies in three animal species and point to gelsolin as a candidate suppressor of breast cancer.

Frequent ectopic expression of a placenta-specific gene at high levels in BALB/c mouse mammary carcinomas.

A placenta-specific gene, MIPP, is transcriptionally regulated in BALB/c mice by a solo long terminal repeat (LTR) of an intracisternal A-particle (IAP), an endogenous retrotransposon. Expression of IAPs, which is also promoted by LTR sequences, is a frequent aberration in many mouse mammary tumors of BALB/c mice. Given that these retroelements and the placental gene have a common promoter, we hypothesized that the tumors also express the gene. Northern blot analysis and RT-PCR revealed high expression of the placenta-specific gene in BALB/c mouse mammary preneoplasias and carcinomas of diverse etiologies, but not in normal mammary gland from virgin, pregnant and lactating mice. The preneoplasias and tumors expressed two transcripts, one of which is apparently unique to the mammary lesions. The other transcript is the same as one expressed in placenta that is not promoted by the IAP LTR. Despite the parallel expression of the placental gene and IAPs in the mammary tissues, RT-PCR showed that LTR sequences are absent from tumor-associated MIPP transcripts. Southern analysis revealed no gross mutations of the MIPP gene in mammary preneoplasias and tumors. The ectopic expression of the placenta-specific gene in BALB/c mouse mammary preneoplasias and carcinomas raises the possibility that it acts as an oncogene.

Comparative expression of the LINE-1 p40 protein in human breast carcinomas and normal breast tissues.

Human LINE-1 (L1Hs) retrotransposons can act as insertional mutagens and are expressed in a variety of tumors, including breast cancer. The purpose of the present study was to examine expression of the p40 protein encoded by the first open reading frame of a L1Hs element in normal human breast tissue of patients without malignant breast disease and in nontumor breast tissue adjacent to cancer and to compare it to expression in breast carcinomas. An antiserum specific for the L1Hs p40 protein was used to analyze its expression in 5 reduction mammoplasties, 16 primary breast cancers, 1 lymph node metastasis and 13 non-malignant breast tissues adjacent to matched primaries by western blotting and/or immunocytochemistry. The immunoreactive band observed on westerns consistently had a M(r) of approximately 46 kDa. Westerns detected some p40 protein expression in all malignant and nonmalignant tissues examined, although 4 of 5 reduction mammoplasties had very low or trace levels as compared with tumors. Nonmalignant breast tissues adjacent to cancers showed significant western band reactivity, and all 15 tumors were positive. Immunocytochemistry revealed staining specificity of the antibody for epithelial cells. Of 12 invasive carcinomas examined, 100% were positive for the p40 protein, whereas one reduction with benign proliferative disease was very weakly reactive, two histologically normal reductions were negative, and 4 of 6 nonmalignant tissues adjacent to cancers were negative. Our data indicated that expression of the L1Hs p40 protein was often elevated in tumor cells of human breast cancers compared to epithelium of normal mammary gland.

Tumor viruses and endogenous retrotransposons in mammary tumorigenesis.

Much of our knowledge about the intricate pathways and molecular mechanisms involved in the conversion of a normal mammary epithelial cell to malignancy derives from studies on mammary tumorigenesis induced by the retrovirus mouse mammary tumor virus. In addition, three DNA tumor viruses, simian virus 40, polyomavirus, and human papillomavirus, have been instrumental in dissecting the series of steps comprising neoplastic progression of mammary epithelium, particularly with cultured human breast cells. Endogenous transposons are analogous bioagents receiving increased attention recently. At least 10% of the cell genome consists of transposable elements, a growing number of which have been implicated in mutagenizing DNA in a variety of human tissues and disorders. Research efforts have therefore intensified to determine if endogenous elements such as retrotransposons participate in the development of breast cancer in animals and humans.

Mutations involving the endogenous ecotropic murine leukemia virus in primary mammary carcinomas of BALB/c mice.

Endogenous murine leukemia virus-related elements (MLVEs) are often overexpressed in primary mammary carcinomas of BALB/c mice. We therefore searched for mutations associated with MLVEs and found amplified sequences of the ecotropic MLVE in hormonally and chemically induced mammary neoplasms. Restriction fragment length polymorphism (RFLP) analysis revealed DNA rearrangements consistent with 1-10 or more new copies of the ecotropic MLVE in the genome of these tumors. This is the first evidence of mutations involving an endogenous retrovirus other than mouse mammary tumor virus in mouse mammary carcinomas.

Cytokeratin expression in human respiratory epithelium of nasal polyps and turbinates.

The cytokertatins in respiratory epithelial cells (REC) of human nasal polyps and turbinates were analyzed by immunohistochemistry. Cytokeratin 19 (CK19) was present in all REC, CK5 and 14 were expressed primarily in basal cells, and CK7, 8, and 18 were found in suprabasal cells. Differences in cytoplasmic locations were also apparent among the individual cytokeratins. CK13 was not detected in any REC of these tissues. The results indicate the profile of cytokeratins in REC of human nasal polyps and turbinates is essentially identical to that of REC in the more distal respiratory tract.

Inhibition of growth of human ovarian cancer in nude mice by luteinizing hormone-releasing hormone antagonist Cetrorelix (SB-75).

OBJECTIVE: To report on the in vitro and in vivo inhibitory effects of LH-releasing hormone (LH-RH) antagonist Cetrorelix (SB-75; Asta Medica, Frankfurt-Main, Germany) against a panel of human ovarian carcinomas. IN VITRO STUDIES: the effect of SB-75 was measured using a standardized chemosensitivity assay in the following ovarian cancer cell lines: UCI 101; UCI 107; PA-1; NIH: OVCAR 3; UCLA: 222; A2780, parental; A2780-CR, cisplatin resistant; A2780-DR, doxorubicin resistant; and the human breast cancer cell line, MCF-7. Results were expressed as percent growth inhibition determined by crystal violet photometric analysis. In vivo studies: the antiproliferative effect of this agent was examined using UCI-107, a primary epithelial ovarian carcinoma cell line, in a nude mouse model. On day 0, 10 x 10(6) UCI 107 cells were implanted subcutaneously into 20 intact female athymic nude mice (5 to 6 weeks old). On day 8, the mice were randomly divided into two groups of 10; control mice were implanted with miniosmotic pumps filled with a vehicle solution consisting of 5.2% mannitol in saline; and treated animals received pumps filled to deliver continuous administration of SB-75 at 60 micrograms per mouse per day. IN VITRO STUDIES: direct inhibition of cell proliferation by SB-75 was not observed at concentrations ranging from 1 nM to 100 microM (exposure lasting three to four cell doublings) with the exception of MCF-7, which demonstrated a 33% inhibition at the latter concentration. In vivo studies: on day 16, caliper measurements were taken from subcutaneous tumor nodules in SB-75-treated and untreated mice and a significant difference of 270% in mean tumor volume was observed. End point was determined, on day 30, when control tumor volume approached 10,000 mm3. At that time the difference in mean tumor volumes increased to 600%, indicating a substantial antiproliferative effect had been achieved in the SB-75-treated group. CONCLUSION: Our in vitro findings show direct inhibition by SB-75 on proliferation of human breast cancer cells. This direct inhibition in vitro was not observed in our ovarian cancer cell lines. However, in vivo SB-75 caused a significant inhibition of growth of human epithelial ovarian cancer. This may be a result of inhibition of the pituitary gonadal axis and gonadotropin secretion. Our results warrant further investigation.

Comparative expression of endogenous retrotransposons in adult mouse mammary gland during normal development and differentiation.

Amplified expression of the endogenous retrotransposons, intracisternal A particles (IAPs) and murine leukemia virus-related elements (MLVEs), along with decreased expression of VL30 elements frequently occurs during mouse mammary tumorigenesis. We have now analyzed the expression of these retroelements during the normal developmental and differentiation cycle of the mammary gland as found in virgin, pregnant, lactating, and postlactation adult female BALB/c mice. Retrotransposon expression was either unchanged or decreased during the progressive stages of the cycle compared to virgin tissue. Likewise, growth of mammary epithelial cells in primary culture had little or no effect on expression of IAPs, MLVEs and VL30 sequences. Thus, the dramatic changes involving these retrotransposons in many mouse mammary tumors appear unrelated to any normal state.