The BET inhibitor JQ1 selectively impairs tumour response to hypoxia and downregulates CA9 and angiogenesis in triple negative breast cancer.

The availability of bromodomain and extra-terminal inhibitors (BETi) has enabled translational epigenetic studies in cancer. BET proteins regulate transcription by selectively recognizing acetylated lysine residues on chromatin. BETi compete with this process leading to both downregulation and upregulation of gene expression. Hypoxia enables progression of triple negative breast cancer (TNBC), the most aggressive form of breast cancer, partly by driving metabolic adaptation, angiogenesis and metastasis through upregulation of hypoxia-regulated genes (for example, carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A). Responses to hypoxia can be mediated epigenetically, thus we investigated whether BETi JQ1 could impair the TNBC response induced by hypoxia and exert anti-tumour effects. JQ1 significantly modulated 44% of hypoxia-induced genes, of which two-thirds were downregulated including CA9 and VEGF-A. JQ1 prevented HIF binding to the hypoxia response element in CA9 promoter, but did not alter HIF expression or activity, suggesting some HIF targets are BET-dependent. JQ1 reduced TNBC growth in vitro and in vivo and inhibited xenograft vascularization. These findings identify that BETi dually targets angiogenesis and the hypoxic response, an effective combination at reducing tumour growth in preclinical studies.

Crohn's disease with gastroduodenal involvement: Diagnostic approach.

Crohn's disease (CD) is a chronic idiopathic inflammatory disease of gastrointestinal tract characterized by segmental and transmural involvement of gastrointestinal tract. Ileocolonic and colonic/anorectal is a most common and account for 40% of cases and involvement of small intestine is about 30%. Isolated involvement of stomach is an extremely unusual presentation of the disease accounting for less than 0.07% of all gastrointestinal CD. To date there are only a few documented case reports of adults with isolated gastric CD and no reports in the pediatric population. The diagnosis is difficult to establish in such cases with atypical presentation. In the absence of any other source of disease and in the presence of nonspecific upper gastrointestinal endoscopy and histological findings, serological testing can play a vital role in the diagnosis of atypical CD. Recent studies have suggested that perinuclear anti-neutrophil cytoplasmic antibody and anti-Saccharomycescervisia antibody may be used as additional diagnostic tools. The effectiveness of infliximab in isolated gastric CD is limited to only a few case reports of adult patients and the long-term outcome is unknown.

An interesting pathological diagnosis - adenoid cystic carcinoma in an adolescent girl.

Adenoid cystic carcinomas (ACCs) constitute 0.1-1 % of all malignant breast tumors. They have better prognosis than other breast malignancies. To date, there have been about 933 cases reported as per English literature. To the best of our knowledge, this case may be the second well-documented case of ACC of breast at younger age.

Microscopic colitis: Common cause of unexplained nonbloody diarrhea.

Microscopic colitis (MC) is characterized by chronic, watery, secretory diarrhea, with a normal or near normal gross appearance of the colonic mucosa. Biopsy is diagnostic and usually reveals either lymphocytic colitis or collagenous colitis. The symptoms of collagenous colitis appear most commonly in the sixth decade. Patients report watery, nonbloody diarrhea of a chronic, intermittent or chronic recurrent course. With collagenous colitis, the major microscopic characteristic is a thickened collagen layer beneath the colonic mucosa, and with lymphocytic colitis, an increased number of intraepithelial lymphocytes. Histological workup can confirm a diagnosis of MC and distinguish the two distinct histological forms, namely, collagenous and lymphocytic colitis. Presently, both forms are diagnosed and treated in the same way; thus, the description of the two forms is not of clinical value although this may change in the future. Since microscopic colitis was first described in 1976 and only recently recognized as a common cause of diarrhea, many practicing physicians may not be aware of this entity. In this review, we outline the epidemiology, risk factors associated with MC, its etiopathogenesis, the approach to diagnosis and the management of these individuals.

Unusual case of insitu (intracystic) papillary carcinoma of breast.

The term "intracystic papillary ductal carcinoma in situ", has recently changed and is now more appropriately referred to as "intracystic papillary carcinoma'' constituting only 0.5% to 1% of all breast cancers. Herein, we discuss an unusual case of intracystic insitu papillary carcinoma of breast in a postmenopausal woman, the diagnosis of which was made on histopathology and confirmed by immunohistochemistry. Patient responded well to postoperative adjuvant radiotherapy without any recurrence, thereby preventing further morbidity and mortality related to invasion or tumor progression. So careful histopathological evaluation is the mainstay to arrive at the correct diagnosis to avoid untoward complications related to under diagnosis and /over diagnosis.

A chronological expression profile of gene activity during embryonic mouse brain development.

The brain is a functionally complex organ, the patterning and development of which are key to adult health. To help elucidate the genetic networks underlying mammalian brain patterning, we conducted detailed transcriptional profiling during embryonic development of the mouse brain. A total of 2,400 genes were identified as showing differential expression between three developmental stages. Analysis of the data identified nine gene clusters to demonstrate analogous expression profiles. A significant group of novel genes of as yet undiscovered biological function were detected as being potentially relevant to brain development and function, in addition to genes that have previously identified roles in the brain. Furthermore, analysis for genes that display asymmetric expression between the left and right brain hemispheres during development revealed 35 genes as putatively asymmetric from a combined data set. Our data constitute a valuable new resource for neuroscience and neurodevelopment, exposing possible functional associations between genes, including novel loci, and encouraging their further investigation in human neurological and behavioural disorders.

Effects of FSH on testicular mRNA transcript levels in the hypogonadal mouse.

FSH acts through the Sertoli cell to ensure normal testicular development and function. To identify transcriptional mechanisms through which FSH acts in the testis, we have treated gonadotrophin-deficient hypogonadal (hpg) mice with recombinant FSH and measured changes in testicular transcript levels using microarrays and real-time PCR 12, 24 and 72 h after the start of treatment. Approximately 400 transcripts were significantly altered at each time point by FSH treatment. At 12 h, there was a clear increase in the levels of a number of known Sertoli cell transcripts (e.g. Fabp5, Lgals1, Tesc, Scara5, Aqp5). Additionally, levels of Leydig cell transcripts were also markedly increased (e.g. Ren1, Cyp17a1, Akr1b7, Star, Nr4a1). This was associated with a small but significant rise in testosterone at 24 and 72 h. At 24 h, androgen-dependent Sertoli cell transcripts were up-regulated (e.g. Rhox5, Drd4, Spinlw1, Tubb3 and Tsx) and this trend continued up to 72 h. By contrast with the somatic cells, only five germ cell transcripts (Dkkl1, Hdc, Pou5f1, Zfp541 and 1700021K02Rik) were altered by FSH within the time-course of the experiment. Analysis of canonical pathways showed that FSH induced a general decline in transcripts related to formation and regulation of tight junctions. Results show that FSH acts directly and indirectly to induce rapid changes in Sertoli cell and Leydig cell transcript levels in the hpg mouse but that effects on germ cell development must occur over a longer time-span.

Protein expression changes in spinal muscular atrophy revealed with a novel antibody array technology.

Autosomal recessive proximal spinal muscular atrophy (SMA) is a severe neurodegenerative disease of childhood causing weakness and wasting secondary to motor neuron dysfunction. Over 97% of cases are caused by deletions or mutations within the survival motor neuron (SMN) gene. The SMN protein is highly expressed within brain, spinal cord and muscle, and is decreased in SMA patients. It has been shown to have an important role in RNA metabolism, but the reason for the specific motor neuron loss is still unclear. We have used a novel antibody array technology to look for differences in the expression patterns of primary muscle cultures from a type II SMA patient and a normal control. A relatively small number of differences were found within a group of proteins that function as both RNA binding proteins and transcription factors. Interactions between a number of these proteins are well established, and three of them bind in turn to p53 which interacts with SMN. A number of the changes were confirmed with western blot analysis both in the primary muscle cultures and in skeletal muscle samples from SMA patients and controls. Changes at the mRNA level were also confirmed with oligonucleotide arrays. These results suggest that a common transcription pathway may be altered in the disease state, and suggests that down-regulation of transcription factors contributes to SMA pathogenesis.

Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery.

In this report it is demonstrated for the first time that rabies-G envelope of the rabies virus is sufficient to confer retrograde axonal transport to a heterologous virus/vector. After delivery of rabies-G pseudotyped equine infectious anaemia virus (EIAV) based vectors encoding a marker gene to the rat striatum, neurons in regions distal from but projecting to the injection site, such as the dopaminergic neurons of the substantia nigra pars compacta, become transduced. This retrograde transport to appropriate distal neurons was also demonstrated after delivery to substantia nigra, hippocampus and spinal cord and did not occur when vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors were delivered to these sites. In addition, peripheral administration of rabies-G pseudotyped vectors to the rat gastrocnemius muscle leads to gene transfer in motoneurons of lumbar spinal cord. In contrast the same vector pseudotyped with VSV-G transduced muscle cells surrounding the injection site, but did not result in expression in any cells in the spinal cord. Long-term expression was observed after gene transfer in the nervous system and a minimal immune response which, together with the possibility of non-invasive administration, greatly extends the utility of lentiviral vectors for gene therapy of human neurological disease.

Direct retroviral delivery of human cytochrome P450 2B6 for gene-directed enzyme prodrug therapy of cancer.

Human cytochrome P450 2B6 (CYP2B6) metabolizes the prodrug cyclophosphamide (CPA) to produce phosphoramide mustard that cross-links DNA leading to cell death. We have constructed a novel retroviral vector encoding CYP2B6 (designated "MetXia-P450") and used it to transduce the human tumor cell lines HT29 and T47D. MetXia-P450 transduction sensitised these cells to the cytotoxic effects of the prodrug CPA. Results from in vitro experiments demonstrated adverse effects on the clonogenic survival of cyclophosphamide-treated cells transduced with MetXia-P450. Cytotoxic activity accompanied by bystander effect was particularly evident in 3-D multicellular spheroid models suggesting that this in vitro system may be a more appropriate model for assessing the efficacy of gene directed-enzyme prodrug therapy (GDEPT). We have applied this approach in a clinically relevant gene therapy protocol on established subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450-based GDEPT strategy mediated by a direct retroviral gene transfer in vivo.

Analysis of 4070A envelope levels in retroviral preparations and effect on target cell transduction efficiency.

A number of stable producer cell lines for high-titer Mo-MuLV vectors have been constructed. Development has previously centered on increasing end-point titers by producing maximal levels of Mo-MuLV Gag/Pol, envelope glycoproteins, and retroviral RNA genomes. We describe the production yields and transduction efficiency characteristics of two Mo-MuLV packaging cell lines, FLYA13 and TEFLYA. Although they both produce 4070A-pseudotyped retroviral vectors reproducibly at >1 x 10(6) LFU ml(-1), the transduction efficiency of unconcentrated and concentrated virus from FLYA13 lines is poor compared with vector preparations from TEFLYA lines. A powerful inhibitor of retroviral transduction is secreted by FLYA13 packaging cells. We show that the inhibitory factor does not affect transduction of target cells by RD114-pseudotyped vectors. This suggests that the inhibitory factor functions at the level of envelope-receptor interactions. Phosphate starvation of target cells shows a two-fold increase in Pit2 receptor mRNA and causes some improvement in FLYA13 virus transduction efficiency. Western blots show that FLYA13 viral samples contain an eight-fold higher ratio of 4070A envelope to p30gag than that of virus produced by TEFLYA producer cell lines. This study correlates overexpression of 4070A envelope glycoprotein in retroviral preparations with a reduction of transduction efficiency at high multiplicities of infection. We suggest that TEFLYA packaging cells express preferable levels of 4070A compared with FLYA13, which not only enables high-titer stocks to be generated, but also facilitates a high efficiency of transduction of target cells.

Quantitative analysis of vascular endothelial growth factor expression in chronic lymphocytic leukaemia.

Vascular endothelial growth factor (VEGF) Flays a central role in solid tumour angiogenesis, although its expression has never been previously documented in circulating lymphoma or leukaemia cells. Here we have quantified mRNA encoding VEGF isoforms 121 and 165 in peripheral lymphocytes of 6/6 CLL patients using Northern blotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR) techniques. No VEGF mRNA could be detected in control lymphocytes. VEGF expression in primary lymphoma cells was lower than in normoxic human colorectal carcinoma cells (LS174T), implying that VEGF expression within tumour cells must be regulated by a range of malignancy-associated factors.

Melanoma cell lines express VEGF receptor KDR and respond to exogenously added VEGF.

Tumour-secreted vascular endothelial growth factor (VEGF) exerts a number of effects which are important in tumour pathology, including stimulation of angiogenesis and permeabilisation of tumour-associated vasculature. In this study we have examined the possibility that VEGF may also play an autocrine role in tumour growth. Using reverse-transcriptase polymerase chain reaction (RT-PCR), the expression of VEGF was found in 15/15 human tumour cell lines examined, while the VEGF receptor KDR was detected only in three melanoma cell lines (MeWo and A375, both wild type and metastatic variant). Exogenously added VEGF (10ng/ml) was able to stimulate up to 40% increased proliferation of A375 M melanoma cells following a 48-h period of quiescence, suggesting that VEGF may indeed play a role in autocrine, as well as paracrine, stimulation of melanoma growth.

Effects of inoculation site and Matrigel on growth and metastasis of human breast cancer cells.

The co-injection of extracellular matrix components, such as Matrigel, with human tumour cells into nude mice has been reported to facilitate tumour formation and growth, but it is unknown whether such components exert similar effects on tumour progression and metastasis. Metastatic behaviour is known to be enhanced when tumour cells are implanted orthotopically, and it is inferred that full and efficient expression of this phenotype may involve some interactions with local connective tissue matrix. It was therefore decided to investigate whether manipulation of the mesenchymal environment by co-injection of extracellular matrix components, in the form of Matrigel, with human breast cancer cells into orthotopic or ectopic sites could augment their metastatic performance, as well as their growth at the site of inoculation. Standard aliquots of 10(6) cells of the polyclonal human breast carcinoma cell line MDA-MB-435, and of four clonal cell lines, two metastatic and two non-metastatic derived from it, were injected with and without Matrigel, orthotopically or subcutaneously into nude mice. The latent period of tumour formation at the inoculation site as well as final tumour size and metastatic performance at autopsy, 140 days after inoculation, were then assessed. The prevalence of metastasis of the parent, polyclonal, cell line and of its metastatic clones was increased if the cell inoculum was mixed with Matrigel. Non-metastatic clones were not induced to become metastatic by this treatment, but local tumour growth at the site of inoculation was enhanced in all experimental groups receiving Matrigel. Orthotopic inoculation acted synergistically with Matrigel to maximise both tumour growth and metastatic behaviour. The composition of the local extracellular matrix at the site of tumour growth influenced expression of the metastatic phenotype by cells which are constitutionally capable of this behaviour, but did not induce it in ones which are not. Previous reports that local tumour growth is facilitated by enrichment of the mesenchymal matrix are confirmed. The mechanisms by which such effects are exerted are worthy of study, to ascertain whether they might be subject to clinical manipulation designed to retard tumour growth and dissemination.

Studies on relationships between metastatic and non-metastatic tumor cell populations using lineages labeled with dominant selectable genetic markers.

The relationships between metastatic and non-metastatic cell populations co-existing in composite neoplasms have been studied using cell lineages marked with a dominant selectable marker (neomycin resistance), by transfection. The experimental circumstances were arranged so that the lineages were known to be genotypically distinct (i.e. not merely phenotypic variants of the same lineage) and so that a single metastatic clone was each time combined with a mixed polyclonal non-metastatic population and both partners were distinctly and recognizably marked. This made it possible to ascertain the fates of clones with different metastatic capabilities during tumor progression and metastasis and evaluate their relative contributions to the clinical extent of disease. It was found that metastatic and non-metastatic cell lineages co-existed in most of the late-stage primary tumors examined and that a cell lineage that is invariably non-metastatic, when growing on its own, can with surprising frequency be found thriving in distant metastatic deposits, when it grows to form a primary tumor in combination with a metastatic partner. In fact, occasional metastases from such tumors contained no detectable cells of the metastatic lineage. The endowment of a tumor cell lineage with a new, clinically significant, capability which it convincingly and reproducibly did not manifest before, by another coexisting cell population raises several new questions about the contribution of such phenomena to the overall debilitating properties of the neoplasm and the geometric progression of its impact on the host.

Correlation of VLA-4 integrin expression with metastatic potential in various human tumour cell lines.

This investigation has focused on whether a number of molecular species, which have recently been recognised as components of cell attachment receptors utilised in recirculatory leukocyte traffic, are expressed on metastatic tumour cell populations. This has been studied on live cultured metastatic and non-metastatic tumour cell lines as well as on histological sections of frozen tissue from primary tumours and metastases which they formed after inoculation into nude mice. Here we report data we have obtained using immunofluorescence microscopy, fluorescence activated cell analysis, immunocytochemistry and pathological investigation of tumour behaviour in vivo, which converge to indicate that expression of the integrin molecule VLA-4 is positively associated with the execution of the metastatic process. This molecule is known to be a receptor for at least two ligands, namely the inducible endothelial adhesion molecule VCAM-1 and the extracellular matrix component fibronectin, and is thought to be mechanistically important in the attachment and diapodesis of lymphocytes. The present findings, indicating differential expression of this molecule on metastatic cell populations relative to non-metastatic cell populations, support and extend recent reports from other laboratories, of the presence of various leukocyte adhesion receptors on metastatic tumour cells. This accumulating evidence suggests that inappropriate expression of one or more of these surface adhesion molecules in tumour cell lineages may endow the progeny of the affected clones with some of the properties needed for metastatic behaviour. The total information so far assembled by various groups also provides some early clues suggesting that the types of molecules expressed may be related to the histogenetic origin of the tumour and its pattern of metastatic spread.(ABSTRACT TRUNCATED AT 250 WORDS)

Fate of clonal lineages during neoplasia and metastasis studied with an incorporated genetic marker.

The fate of clonal lineages in tumor formation and metastasis has been studied by genotypic marking of cells from three separate tumor lines of different malignant potential. Marking was accomplished by random incorporation of the neomycin resistance gene and visualized by Southern blot analysis of integration sites. Primary tumors formed by polyclonal cell suspensions of all three cell lines injected s.c. usually remained polyclonal even at late stages of tumor growth and metastatic spread. Lung metastases were often clonal, but it was not unusual to find ones of polyclonal origin. Lymph node metastases were almost always polyclonal and remained so, as they grew large. Sometimes clones present in the original inoculum were absent in the primary. Other times clones visible in the metastases were undetectable in the corresponding primary tumor. Occasionally a single clone became dominant in the primary, and others were eliminated, but this was not a necessary prelude to the onset of invasive or metastatic behavior. It is concluded that there is considerable variation in the results obtained with various cell lines in different circumstances. Even clones which are underrepresented in the original inoculum or the primary tumor can acquire metastatic capability. Hence, progression of malignancy is not uniformly dependent on prior or concurrent extinction of other non- or less metastatic clones in the neoplasm, and the underlying mechanisms of invasion and metastasis can be separated from those which sometimes confer growth supremacy on a clone of tumor cells. The frequent continuing genetic heterogeneity of cells in a neoplasm has substantial implications for clinical treatment protocols.