MMTV-like sequences in human breast cancer: a fluorescent PCR/laser microdissection approach

The hypothesis that a retrovirus homologous to the mouse mammary tumour virus (MMTV) is involved in human breast cancer aetiology has fascinated scientists from many years, but it has never been convincingly demonstrated. Renewed interest in this hypothesis developed when an MMTV env gene-like sequence was found in 38% of human breast cancer tissues. Whereas some subsequent studies confirmed these findings, others did not. The main reasons for this discrepancy, among others, are the different sensitivities and technical details of current molecular approaches to the detection of these sequences. This study is an attempt to find sensitive and reproducible conditions capable of detecting MMTV env-like sequence in human samples. To this end, we first developed a fluorescence nested-PCR (FN-PCR) method that was able to detect very low copies of the viral genome, and then screened a panel of 45 frozen breast cancer samples obtained by laser microdissection. The MMTV env gene-like sequence was found in 15 (33%) of the human breast cancers analysed, whereas the same sequence was detectable neither in normal tissues nor in other types of tumour. Sequence analysis revealed 96% homology with the MMTV genome, but no other significant similarities with the human genome. The combined use of frozen material, microdissected cell populations and FN-PCR provides a novel, sensitive, robust, non-radioactive and fast methodology for the molecular detection of human-MTV. This approach might be successfully used in large molecular studies that aim to investigate the hypothesis of a retroviral aetiology of human breast cancer.     

Presence of mouse mammary tumour-like virus gene sequences may be associated with morphology of specific human breast cancer

BACKGROUND: Mouse mammary tumour virus (MMTV) has a proven role in breast carcinogenesis in wild mice and genetically susceptible in-bred mice. MMTV-like env gene sequences, which indicate the presence of a replication-competent MMTV-like virus, have been identified in some human breast cancers, but rarely in normal breast tissues. However, no evidence for a causal role of an MMTV-like virus in human breast cancer has emerged, although there are precedents for associations between specific histological characteristics of human cancers and the presence of oncogenic viruses. AIM: To investigate the possibility of an association between breast cancer and MMTV-like viruses. METHODS: Histological characteristics of invasive ductal human breast cancer specimens were compared with archival MMTV-associated mammary tumours from C3H experimental mice. The presence of MMTV-like env DNA sequences in the human breast cancer specimens was determined by polymerase chain reaction and confirmed by Southern hybridisation. RESULTS: MMTV-like env gene sequences were identified in 22 of 59 (37.3%) human breast cancer specimens. Seventeen of 43 (39.5%) invasive ductal carcinoma breast cancer specimens and 4 of 16 (25%) ductal carcinoma in situ specimens had some histological characteristics, which were similar to MMTV-associated mouse mammary tumours. However, these similarities were not associated with the presence or absence of MMTV-like gene sequences in the human breast tumour specimens. A significant (p = 0.05) correlation was found between the grade of the human breast cancer and similarity to the mouse mammary tumours. The lower the grade, the greater the similarity. CONCLUSION: Some human breast cancer specimens, in which MMTV-like env DNA sequences have been identified, were shown to have histological characteristics (morphology) similar to MMTV-associated mouse mammary tumours. These observations are compatible with, but not conclusive of, an association between the presence of MMTV-like env DNA sequences and some human breast cancers.     

Correlation of partial env gene sequences with disease progression parameters in HIV-positive pregnant women from India

Ever since the beginning of the epidemic of HIV, one of the poignant aspects of HIV infection is transmission of the virus from mother to child. It is not known whether pregnancy accelerates the progression of HIV infection from a clinically asymptomatic stage to a progressive clinical phase. Present study was carried out to understand disease progression in pregnant women from India. We studied co-receptor utilization (the major determinant of HIV disease progression), N-glycosylation sites, and sequence variability. Blood samples were collected from 25 HIV sero-positive patients, eleven from the antenatal risk group (experimental group), nine from heterosexual male, and five from heterosexual female risk group (control group). Partial env gene was amplified by PCR and sequenced. BLAST search and phylogenetic analysis were used to determine the subtype. The deduced amino acid sequence of the V3 region was used to predict co-receptor, determine sequence variability and N-glycosylation site. The experimental group comprising the antenatal risk group did not exhibit any difference in terms of co-receptor, N-glycosylation, and sequence variability when compared with the control, non-pregnant group. Pregnancy does not seem to accelerate the clinical course of HIV infection. The female body during the gestation phase possibly acquires certain strategies to impede or at least alleviate the disease progression during the crucial immune-compromised pregnancy phase, which would otherwise adversely affect the mother as well as the fetus during the infection.     

De novo retrotransposition of unbiased sequences in a human breast cancer cell clone.

It has been demonstrated recently that certain repetitive sequences and even expressed single-copy genes are capable of retrotransposition, but little is known about the endogenous or exogenous modifiers of this process in human cells. Retrotransposition may contribute to gene inactivation and genetic instability in cancer development. We have used the human cell line MCF-7 to generate a method for investigating de novo retrotransposition in breast cancer cells. The strategy employs a reporter construct transfected into MCF-7 cells that encodes neomycin phosphotransferase gene (neoR) sequences interrupted by an intron derived from the gamma-globin gene and sandwiched between two promoters in opposite orientation; the phosphotransferase is not produced in transfected cells expressing the plasmid until transposition via a spliced antisense neoR RNA intermediate has occurred, conferring a functional gene product and thereby resistance to G418. A stable transfectant line that showed presence of reporter plasmid DNA and expression of reporter antisense neoR was obtained and used to demonstrate spontaneous retrotransposition of neoR sequences: tester cells were subjected to selection in G418 medium, and neomycin-resistant clones were isolated at a frequency of 10(-7). A simple PCR-based prescreening of colonies fixed and stained in Petri dishes can be used to verify intronless neoR DNA. Expanded populations of G418-resistant colonies were determined to be derived from reporter sequences that had transposed via an RNA intermediate by Southern blot genotyping. This experimental assay may be used for exploring endogenous and environmental factors that influence host cell-mediated retrotransposition of unbiased cellular sequences in breast tumor cells. Genes Chromosomes Cancer 26:84-91, 1999.     

人乳腺癌细胞化疗敏感性与相关基因表达的关系

目的探讨人乳腺癌细胞化疗敏感性与多药耐药相关基因和凋亡调控基因表达的关系。方法MTT法检测5株人乳腺癌细胞株Bcap37、MCF-7、T47D、MDA-MB-231和MDA-MB-435对阿霉素(ADM)、顺铂(DDP)、丝裂霉素(MMC)、5-氟尿嘧啶(5-Fu)、卡氮芥(BCNU)的敏感性。流式细胞术检测多药耐药基因P-糖蛋白(P-GP)、谷胱甘肽-S-转移酶-π(GST-π)、肺耐药蛋白(LRP)、多药耐药相关蛋白(MRP)和甲基鸟嘌呤甲基转移酶(MGMT)等耐药相关基因和凋亡调控基因FAS、BCL-2、P53和P16的表达。结果不同人乳腺癌细胞株对同一药物敏感性差异较大。相关分析表明,细胞株对5-Fu的敏感性与P16表达呈正相关(P<0.01),对其余药物的敏感性与所检基因的表达水平无关。结论人乳腺癌细胞株对5-Fu的敏感性与P16表达有关。     

Characterization of human MMTV-like (HML) elements similar to a sequence that was highly expressed in a human breast cancer: further definition of the HML-6 group

Previously, we found a retroviral sequence, HML-6.2BC1, to be expressed at high levels in a multifocal ductal breast cancer from a 41-year-old woman who also developed ovarian carcinoma. The sequence of a human genomic clone (HML-6.28) selected by high-stringency hybridization with HML-6.2BC1 is reported here. It was 99% identical to HML-6.2BC1 and gave the same restriction fragments as total DNA. HML-6.28 is a 4.7-kb provirus with a 5'LTR, truncated in RT. Data from two similar genomic clones and sequences found in GenBank are also reported. Overlaps between them gave a rather complete picture of the HML-6.2BC1-like human endogenous retroviral elements. Work with somatic cell hybrids and FISH localized HML-6.28 to chromosome 6, band p21, close to the MHC region. The causal role of HML-6.28 in breast cancer remains unclear. Nevertheless, the ca. 20 Myr old HML-6 sequences enabled the definition of common and unique features of type A, B, and D (ABD) retroviruses. In Gag, HML-6 has no intervening sequences between matrix and capsid proteins, unlike extant exogenous ABD viruses, possibly an ancestral feature. Alignment of the dUTPase showed it to be present in all ABD viruses, but gave a phylogenetic tree different from trees made from other ABD genes, indicating a distinct phylogeny of dUTPase. A conserved 24-mer sequence in the amino terminus of some ABD envelope genes suggested a conserved function.     

Cytokine regulation of env gene expression of human endogenous retrovirus-R in human vascular endothelial cells.

To determine whether human endogenous retroviruses are implicated in the pathogenesis of inflammatory vascular diseases of unknown etiology, we examined mRNA expression of a human endogenous retrovirus, HERV-R, which has a long open reading frame in the env region, in cultured human vascular endothelial and smooth muscle cells stimulated in the presence of various cytokines. mRNA of HERV-R was always evident in these cells but not in fibroblastic cells. Levels of expression in vascular endothelial cells were significantly regulated by treatment with tumor necrosis factor-alpha, interleukin (IL)-1alpha, and IL-1beta as up-regulators and interferon-gamma as a down-regulator. These observations are interpreted to mean that HERV-R expression may be up- or down-regulated at sites of inflammation in vessels in vivo and hence may play a pathogenetic role in inflammatory vascular diseases in humans, perhaps similar to endogenous retroviruses in mouse models of polyarteritis nodosa in humans.     

Unique sequences in the env gene of avian hemangioma retrovirus are responsible for cytotoxicity and endothelial cell perturbation

An avian retrovirus isolated from spontaneous cavernous hemangiomas of layer hens codes for an env protein that induces a cytopathic effect on a wide variety of cultured avian and mammalian cells and also causes thrombogenicity of endothelial cells. Sequence analysis of the avian hemangioma inducing virus revealed unique elements in both its env gene and its LTR. We propose that these elements are responsible for the biological and pathogenic characteristics of the virus.     

PTEN gene in triple-negative breast cancer

The paper gives the results of studying the expression of the PTEN gene product by an immunohistochemical method, as well as deletion of the gene by fluorescence in situ hybridization in the tumor cells of 80 patients with triple-negative breast cancer (TNBC). The gene product was absent in the tumor cell nuclei in 56 products. The bulk of the remaining patients who showed a positive response had lobular carcinomas or tumors with an unidentified microscopic variant due to secondary posttherapeutic changes. Lobular carcinomas in a control group presented by patients without TNBC also contained the PTEN gene product. Therefore, all positively responding tumor cells failed to express basaloid markers and androgen receptors. The immunohistochemically detectable absence of the PTEN gene product is usually coupled with deletion at locus 10q23; however, in several cases the negative immunohistochemical reaction is associated with no deletion at the above locus, which suggests that there are mechanisms for PTEN gene dysregulation other than deletion in TNBC. The presence of the PTEN gene product in the tumor cells is associated with good prognosis in patients with TNBC.     

芪三酚与人乳腺癌MDC1基因关系的研究

目的探讨芪三酚（Res）对人乳腺癌MDA—MB-231细胞增殖抑制的相关效应及其与MDCl基因的关系。方法以人乳腺癌MDA—MB-231细胞株为研究对象,采用MTS方法测定细胞增殖,应用吖啶橙荧光染色观察Res对乳腺癌MDA—MB-231细胞的影响,用RT-PCR与免疫印迹方法测定MDCl基因与蛋白表达水平,用小RNA干扰MDCl基因后,用流式细胞仪检测细胞的凋亡并观察其对Res的敏感性影响。结果40μmol／L以上的Res可显著抑制乳腺癌MDA—MB-231细胞的增殖（P〈0．05）,给予0、60、120μmol／LRes能明显降低MDCl基因和蛋白的表达（P〈0．05）。用小RNA干扰MDC1基因后,流式细胞术分析显示,实验组（MDCl．siRNA）的细胞凋亡率[（45．13±6．2）％]较阴性对照组[（24．34±2．6）％]和未处理组[（17．69±4．9）％]明显上升（P〈0．05）,MTS结果显示MDCl基因干扰后细胞对Res的敏感性增加。结论40μmol／L以上的Res可以抑制MDA-MB-231细胞的增殖,Res可以有效降低MDC基因和蛋白的表达并促进细胞的凋亡。用小RNA干扰MDCl基因（MDCl-siRNA）后,MDA-MB-231细胞对Res的敏感性增加。     

Epigenetic inactivation of microRNA gene hsa-mir-9-1 in human breast cancer

MicroRNAs (miRNAs) represent a new class of small non-coding RNAs regulating gene expression by inducing RNA degradation or interfering with translation. Aberrant miRNA expression has been described for several human malignancies and tumour suppressor functions have been ascribed to this new class of small regulatory RNAs. Accordingly, inactivation due to deletion or mutation has been found in human malignancies. Here, we describe the role of aberrant hypermethylation as an additional mechanism for miRNA gene inactivation in human breast cancer. Aberrant hypermethylation was shown for mir-9-1, mir-124a3, mir-148, mir-152, and mir-663 in 34-86% of cases in a series of 71 primary human breast cancer specimens. For comprehensive methylation analysis, combined bisulphite restriction analysis, bisulphite sequencing, and Pyrosequencing were employed. miRNA gene hypermethylation correlated strongly with methylation of known tumour suppressor genes (p = 0.003). After treatment of various breast cancer cell lines with the demethylating agent 5-aza-2'-deoxycytidine, reduction of mir-9-1 gene methylation and concomitant reactivation of expression could be observed. For the mir-9-1 gene, which is already hypermethylated in pre-invasive intraductal lesions, a good correlation between quantitative methylation level and reduction of expression could be demonstrated in a subset of primary human breast cancer specimen (r = 0.8). In conclusion, this study demonstrates that various microRNA genes are also affected by epigenetic inactivation due to aberrant hypermethylation and that this is an early and frequent event in breast cancer development.     

Chromosomal location of human P-glycoprotein gene sequences

Nonresponse to chemotherapy may result from the acquisition of multidrug resistance by malignant cells. Overexpression of the 170,000 dalton cell surface P-glycoprotein is associated with this phenotype and this appears to result from amplification of a multigene family coding for this protein. A cDNA encoding a conserved portion of P-glycoprotein has been cloned from hamster cells, and this was used in the present study to localize human P-glycoprotein gene sequences to chromosome 7q36.     

紫杉醇对乳腺癌细胞紫杉醇耐药基因Txr1表达的影响

目的应用人乳腺癌细胞制备紫杉醇耐药细胞模型,探讨紫杉醇对乳腺癌细胞中紫杉醇耐药基因Txr1表达的影响。方法比较紫杉醇预处理前后不同浓度紫杉醇对乳腺癌细胞的增殖和细胞周期的改变;用RT-PCR方法检测Txr1、TSP1和MDR1的mRNA水平变化;用Western blot检测Txr1和TSP1的蛋白质水平的变化。结果紫杉醇预处理后,紫杉醇抑制乳腺癌细胞(MCF-7细胞)的生长作用明显减弱。流式细胞仪分析表明,MCF-7细胞经过紫杉醇作用后被阻滞于细胞周期的G2M期。紫杉醇作用于MCF-7细胞后,Txr1的mRNA水平上调,TSP1下调,而MDR1表达无明显改变。类似的结果在蛋白的水平被Western blot进一步证实。结论紫杉醇可能通过Txr1上调抑制TSP1的表达从而诱导肿瘤细胞的耐药性。     

乳癌组织端粒酶活性表达及其与p16基因的关系

目的：探讨乳房恶性肿瘤组织中端粒酶活性的表达情况及ｐ１６抑癌基因的影响。方法：应用端粒重复放大程序－酶标法（ＴＲＡＰ－ＥＬＩＳＡ）及ＴＲＡＰ－银染法检测３５例有乳癌组织及２４例良性肿瘤组织中的端粒酶活性,并采用多重ＰＣＲ技术对乳癌组织的ｐ１６基因的缺失空谈进行了分析,结果：３５例乳癌标本中有２８例端粒酶活性表达为阳性（８０％）,而２４例乳房良性肿瘤标本中仅１例端粒酶活性表达阳性（４．１％）。此外３５例乳癌标本中的１５例存在有ｐ１６基因的外显子２及外显子１的缺失（４２．８％）,而该１５例中有１４例被检测为端粒酶活性阳性（９３．３％）,但２０例未缺失标本的端粒酶阳怀率仅为７０％（１４／２０）。结论：端粒酶活性与乳房肿瘤组织的恶性程度密切相关,提示端粒酶参与了肿瘤的形成过程,而端粒酶活性的增高与ｐ１６抑癌基因的缺失突