A map of the human immunoglobulin VH locus completed by analysis of the telomeric region of chromosome 14q.

Analysis of the telomeric region of chromosome 14q has enabled us to complete a map of the immunoglobulin VH locus which accounts for almost all VH segments known to rearrange in B-lymphocytes. The human germline VH repertoire consists of approximately 50 functional VH segments--the exact number depending on the haplotype--spanning 1,100 kilobases upstream of the JH segments. A yeast artificial chromosome used to map these segments was isolated by its ability to provide telomere activity in yeast, suggesting that the VH locus may be located within a few kilobases of the 14q telomere. The limited structural diversity encoded by the functional VH segments demonstrates the importance of combinatorial diversity produced by VDJ joining and the association of heavy and light chains in producing the human antibody repertoire.

Genomic modelling of the ESR1 Y537S mutation for evaluating function and new therapeutic approaches for metastatic breast cancer.

Drugs that inhibit estrogen receptor-α (ER) activity have been highly successful in treating and reducing breast cancer progression in ER-positive disease. However, resistance to these therapies presents a major clinical problem. Recent genetic studies have shown that mutations in the ER gene are found in >20% of tumours that progress on endocrine therapies. Remarkably, the great majority of these mutations localize to just a few amino acids within or near the critical helix 12 region of the ER hormone binding domain, where they are likely to be single allele mutations. Understanding how these mutations impact on ER function is a prerequisite for identifying methods to treat breast cancer patients featuring such mutations. Towards this end, we used CRISPR-Cas9 genome editing to make a single allele knock-in of the most commonly mutated amino acid residue, tyrosine 537, in the estrogen-responsive MCF7 breast cancer cell line. Genomic analyses using RNA-seq and ER ChIP-seq demonstrated that the Y537S mutation promotes constitutive ER activity globally, resulting in estrogen-independent growth. MCF7-Y537S cells were resistant to the anti-estrogen tamoxifen and fulvestrant. Further, we show that the basal transcription factor TFIIH is constitutively recruited by ER-Y537S, resulting in ligand-independent phosphorylation of Serine 118 (Ser118) by the TFIIH kinase, cyclin-dependent kinase (CDK)7. The CDK7 inhibitor, THZ1 prevented Ser118 phosphorylation and inhibited growth of MCF7-Y537S cells. These studies confirm the functional importance of ER mutations in endocrine resistance, demonstrate the utility of knock-in mutational models for investigating alternative therapeutic approaches and highlight CDK7 inhibition as a potential therapy for endocrine-resistant breast cancer mediated by ER mutations.

Inhibitory effects of activin on the growth and morpholgenesis of primary and transformed mammary epithelial cells.

Activin is a member of the transforming growth factor beta superfamily, which is known to have activities involved in regulating differentiation and development. By using reverse transcription-PCR analysis on immunoaffinity-purified human breast cells, we have found that activin beta a and activin type II receptor are expressed by myoepithelial cells, whereas no expression was detected in other breast cell types. In examining 15 breast cell lines, we have found only four (HBL-100, MCF10-A, PMC-42, and BT 20) to be positive for activin beta a mRNA, whereas all expressed the activin type II receptor. Furthermore, we have found activin A to be a potent growth inhibitor of MCF- 7 cells (at 2 ng/ml), where it causes an arrest in G(1). Activin A does not appear to have an effect on the cell cycle of primary myoepithelial or luminal cells. However, we demonstrate that activin is an inhibitor of tubule formation by human mammary organoids in vitro. These are the first observations of activin and activin receptor in the normal human breast and in human breast cell lines and suggest a role for activin in mammary cell growth and morphogenesis.

Loss of cyclin D2 expression in the majority of breast cancers is associated with promoter hypermethylation.

Cyclin D2 is a member of the D-type cyclins, implicated in cell cycle regulation, differentiation, and malignant transformation. It was noted previously that cyclin D2 is not expressed in the majority of breast cancer cell lines, whereas abundant expression was detected in finite life span human mammary epithelial cells. By reverse transcription-PCR and Western blot analysis, we extended this finding to primary breast carcinomas and show that the majority of these tumors lack expression of cyclin D2 mRNA (18 of 24) and protein (10 of 13). In contrast, both luminal and myoepithelial subpopulations of normal breast tissues expressed cyclin D2. Hypermethylation of the CpG island in the promoter was detected by methylation-specific PCR in nearly half of the breast cancers (49 of 106) and was associated with silencing of cyclin D2 gene expression. Promoter hypermethylation was also detected in ductal carcinoma in situ, suggesting that loss of cyclin D2 expression is an early event in tumorigenesis. Our results suggest that loss of cyclin D2 expression is associated with the evolution of breast cancer.

Wilms' tumor suppressor gene (WT1) is expressed in primary breast tumors despite tumor-specific promoter methylation.

We analyzed Wilms' tumor suppressor 1 (WT1) expression and its regulation by promoter methylation in a panel of normal breast epithelial samples and primary carcinomas. Contrary to previous reports, WT1 protein was strongly expressed in primary carcinomas (27 of 31 tumors) but not in normal breast epithelium (1 of 20 samples). Additionally, the WT1 promoter was methylated in 6 of 19 (32%) primary tumors, which nevertheless expressed WT1. The promoter is not methylated in normal epithelium. Thus, although tumor-specific methylation of WT1 is established in primary breast cancer at a low frequency, other transcriptional regulatory mechanisms appear to supercede its effects in these tumors. Our results demonstrate expression of WT1 in mammary neoplasia, and that WT1 may not have a tumor suppressor role in breast cancer.

Expression of FGFR2 BEK and K-SAM mRNA variants in normal and malignant human breast.

The expression of mRNA encoding alternative forms of fibroblast growth factor receptor 2 (FGFR2) differing in the carboxy terminal half of their third immunoglobulin-like domain, was investigated in 77 human breast cancer tissues, 12 non-malignant breast biopsies and 29 cell lines, using a reverse transcriptase (RT) polymerase chain reaction (PCR) method. RNA from the two tissue groups yielded PCR product corresponding to both the BEK and the K-SAM form; amounts normalised to glyceraldehyde phosphate dehydrogenase product were similar in both groups. The level of either variant or of the total FGFR2 product was essentially unrelated to prognosis or clinical status except that patients with advanced clinical T staging had a higher proportion of BEK to K-SAM (P = 0.01). RNA from 1/2 normal breast derived and 8/10 breast cancer cell lines expressed exclusively or predominantly the K-SAM form; 2/10 had significant amounts of both BEK and K-SAM mRNA. Of 12 other epithelial lines, seven expressed mainly K-SAM mRNA, four expressed BEK and one was negative. Of five non-epithelial lines, one was negative, two expressed only BEK mRNA and two had significant amounts of both variants. We conclude that tissue levels of FGFR2 mRNA are unaltered in breast cancer extracts and that the splicing mechanism for this exon selection appears not to be significantly disrupted.

Retroviral infection of the FGF2 gene into MCF-7 cells induces branching morphogenesis, retards cell growth and suppresses tumorigenicity in nude mice.

FGF2 (basic fibroblast growth factor) is a multifunctional growth factor and exhibits diverse function in different cell types. In breast, loss of FGF2 expression is associated with malignant progression. In order to understand the role of FGF2 in maintenance of normal breast structures and control of cell growth, we restored FGF2 expression in the breast cancer cell line MCF-7. The FGF2 retrovirally infected MCF-7 cells (MCF-7.F2.5) not only expressed FGF2 in cytoplasm and nuclei, but also released FGF2 into culture medium both on plastic and in Matrigel conditions. The MCF-7.F2.5 cells formed branches in Matrigel and this effect was abolished by the addition of a neutralising anti-FGF2 antibody or function blocking antibodies to alpha2, alpha3 and beta1 integrins. Furthermore, MCF-7.F2.5 cells lost their ability for anchorage-independent growth in soft agar. When MCF-7 and MCF-7.F2.5 cells were injected into nude mice, there was a 1.6- to 3.2-fold reduction of tumour volume with MCF-7.F2.5 cells in comparison with the parental MCF-7 cells. MCF-7.F2.5 cells also demonstrated a reduction in oestrogen receptor-alpha (ERalpha) both in vitro and in vivo. Our results suggest that introduction of the FGF2 gene into MCF-7 cells altered the malignant tumour cells towards a more benign phenotype in vitro and in vivo.

In vitro regulation of HGF-SF expression by epithelial-mesenchymal interactions.

Studies of parameters which affect cellular proliferation, cellular differentiation together with studies of cell-cell interactions which affect cell behavior are particularly interesting in that they can be used to identify and characterise molecules which through changes in gene expression induce/inhibit cell proliferation, differentiation and movement. Such studies are crucial not only in the context of understanding growth and development, but also in understanding the processes of wound healing and regeneration as well as tumor invasion and metastasis. Here we present a summary of some cell culture models which we have developed for the study of the above-mentioned phenomenon, together with their application to the studies of regulation of HGF-SF expression.

Stable expression of CD1a molecule in human epithelial cell lines shows temperature-dependent expression and affects cell morphology and growth.

The human CD1a molecule is a transmembrane protein which shares structural similarities with HLA class I molecules. It has restricted tissue distribution in normal individuals, and is a useful diagnostic marker for certain disease states such as Langerhans cell histiocytosis. In order to investigate the function of this molecule, a cDNA fragment encoding the CD1a molecule was cloned into several EUKARYOTIC expression vectors which were then used to establish human epithelial cell lines stably expressing the membrane-bound CD1a molecule. Human keratinocytes (HaCaT) and epithelial cells (HeLa) stably expressing CD1a were established by retroviral-mediated gene transfer and DNA transfection, respectively. Expression and localization of the CD1A molecule were then confirmed by Northern blot analysis and immunofluorescence methods. CD1a expression appears to have profound effects on cellular growth and morphology. Both stably CD1a-expressing HeLa and HaCaT cells showed increased doubling times, and up to 20% of CD1a-expressing cells showed altered cell morphology. Clonogenicity experiments demonstrated a reduction in colony size and plating efficiency was augmented in CD1a-positive cells when compared with vector-transfected/infected controls. Our findings suggest that CD1A expression may act as a negative growth regulator in these cells in vitro. Furthermore, lower temperatures greatly enhanced the expression of CD1a at both the protein and mRNA levels in a time-dependent fashion. Since the physiological skin temperatures lie well below the core temperature, this observation may have important implications in the study of Langerhans cells in vitro.

The use of the restriction endonuclease DdeI for mapping relataed DNAs.

Restriction endonuclease mapping of BK virus strain GS using DdeI has allowed us to correct and extend the previously published map.

Conserved gene sequences for species identification: PCR analysis of the 3' UTR of the SON gene distinguishes human and other mammalian DNAs.

In this paper we show that the polymerase chain reaction (PCR) can be used on regions of highly conserved genes, such as the 3' untranslated region (3' UTR) of the SON gene, to identify the mammalian origin of a sample. Using this test, we have been able to distinguish human, monkey, cat, dog, mouse and hamster DNAs. We have also determined the DNA sequence of these different PCR products, which can be used to reinforce species identification. The advantages of this test are that: (i) no prior information is required on the possible species origin of a sample, (ii) the reaction produces a single PCR product which varies in size according to the species of origin, making the test simple to interpret, and (iii) the target region of DNA amplified in these experiments is small and can easily be sequenced and sized using automated techniques. This small size has enabled us to successfully amplify this product from DNA extracted from compromised material (human bone samples) and so demonstrate that the test is valuable for the characterisation of remains in which DNA is degraded.

Capsaicin-sensitive primary sensory neurons in the mouse express N-Acyl phosphatidylethanolamine phospholipase D.

Our previous finding, that the capsaicin- and KCl-induced Ca(2+)-dependent production of the intra- and intercellular signaling molecule N-arachidonoyl ethanolamine (anandamide) in cultured primary sensory neurons could be abolished and reduced by approximately 2/3 by capsaicin-induced degeneration of capsaicin-sensitive neurons, respectively suggests that a major sub-population of capsaicin-sensitive cells together with a group of non-capsaicin-sensitive cells should express enzymes involved in Ca(2+)-dependent anandamide synthesis. N-acyl phosphotidylethanolamine phospholipase D (NAPE-PLD) is known to be involved in Ca(2+)-dependent anandamide production. Hence, here, we used reverse transcriptase and quantitative real time polymerase chain reaction to study NAPE-PLD expression in dorsal root ganglia and to clarify the sub-population of cells expressing this enzyme. Cultures prepared from mouse dorsal root ganglia were grown either in the absence or presence of the neurotoxin, capsaicin (10 muM) overnight. We report, that NAPE-PLD is expressed both in dorsal root ganglia and cultures prepared from dorsal root ganglia and grown in the absence of capsaicin. Furthermore, we also report that capsaicin application downregulates the expression of NAPE-PLD as well as the capsaicin receptor, transient receptor potential vanilloid type 1 ion channel, by about 70% in the cultures prepared from dorsal root ganglia. These findings indicate that a major sub-population of capsaicin-sensitive primary sensory neurons expresses NAPE-PLD, and suggest that NAPE-PLD is expressed predominantly by capsaicin-sensitive neurons in dorsal root ganglia. These data also suggest that NAPE-PLD might be a target to control the activity and excitability of a major sub-population of nociceptive primary sensory neurons.

Microarray coupled to quantitative RT-PCR analysis of androgen-regulated genes in human LNCaP prostate cancer cells.

The androgen receptor (AR) mediates the growth-stimulatory effects of androgens in prostate cancer cells. Identification of androgen-regulated genes in prostate cancer cells is therefore of considerable importance for defining the mechanisms of prostate-cancer development and progression. Although several studies have used microarrays to identify AR-regulated genes in prostate cancer cell lines and in prostate tumours, we present here the results of gene expression microarray profiling of the androgen-responsive LNCaP prostate-cancer cell line treated with R1881 for the identification of androgen-regulated genes. We show that the expression of 319 genes is stimulated by 24 h after R1881 addition, with a similar number (300) of genes being significantly repressed. Expression of the upregulated genes, as well as of 60 of the most robustly downregulated genes, was carried out using quantitative RT-PCR (Q-RT-PCR) over a time-course of R1881 treatment from 0 to 72 h. Q-RT-PCR was also carried out following treatment with other AR agonists (dihydrotestosterone, estradiol and medroxyprogesterone) and antagonists (cyproterone acetate, hydroxyflutamide and bicalutamide). This study provides a comprehensive analysis of androgen-regulated gene expression in the LNCaP prostate cancer cell line, and identifies a number of androgen-regulated genes, not described previously, as candidates for mediating androgen responses in prostate cancer cells.

Inhibiting estrogen responses in breast cancer cells using a fusion protein encoding estrogen receptor-alpha and the transcriptional repressor PLZF.

Estrogen receptor alpha (ERalpha) is a ligand-inducible transcription factor that acts to regulate gene expression by binding to palindromic DNA sequence, known as the estrogen response element, in promoters of estrogen-regulated genes. In breast cancer ERalpha plays a central role, where estrogen-regulated gene expression leads to tumor initiation, growth and survival. As an approach to silencing estrogen-regulated genes, we have studied the activities of a fusion protein between ERalpha and the promyelocytic leukemia zinc-finger (PLZF) protein, a transcriptional repressor that acts through chromatin remodeling. To do this, we have developed lines from the estrogen-responsive MCF-7 breast cancer cell line in which the expression of the fusion protein PLZF-ERalpha is conditionally regulated by tetracycline and shows that these feature long-term silencing of the expression of several well-characterized estrogen-regulated genes, namely pS2, cathepsin-D and the progesterone receptor. However, the estrogen-regulated growth of these cells is not inhibited unless PLZF-ERalpha expression is induced, an observation that we have confirmed both in vitro and in vivo. Taken together, these results show that PLZF-ERalpha is a potent repressor of estrogen-regulated gene expression and could be useful in distinguishing estrogen-regulated genes required for the growth of breast cancer cells.

In vitro regulation of HGF/SF expression.

Studies of parameters which affect cellular proliferation, cellular differentiation and cell-cell interactions influencing cell behaviour are of particular interest. They can be used to identify and characterise molecules which, through changes in gene expression, induce or inhibit cell proliferation, differentiation and movement. Such studies are crucial, not only in the context of understanding growth and development, but also in understanding the processes of wound-healing and regeneration, tumour invasion and metastasis. Here we present a summary of some cell culture models which we have developed for the study of the above-mentioned phenomena, together with their application to studies of the regulation of HGF/SF expression.

A VH gene is located within 95 Kb of the human immunoglobulin heavy chain constant region genes.

Using cosmids covering about 117 Kb upstream of the human immunoglobulin chain C mu gene, we have identified a potentially functional VH gene, belonging to the VHVI subgroup. This VHVI gene is only about 95 Kb from the C mu gene and is probably the first functional VH segment of the Igh locus. These results illustrate the proximity of the human VH, DH and JH segments involved in creation of the complete heavy chain genes.