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Are some breast carcinomas polyclonal in origin?

This editorial comments on the important study by Going et al. published in the present issue of the Journal [1]. Using a molecular genetic assay based on the X-chromosome inactivation principle, they found that 4 out of 12 breast carcinomas examined exhibited what the authors call "clonal mosaicism" that is, two or more monoclonal samples were mosaic (polyclonal) in respect of X chromosome inactivation between separate, morphologically homogeneous tumour areas. The authors very carefully discuss potential methodological errors that could have led to this surprising finding, which seems to run counter to the almost unanimously held conviction that carcinomas are monoclonal in origin, but none of these potential errors would explain the results. As often in such situations, the authors prudently state that further studies using independent analytical techniques are necessary to find out whether a significant proportion of mammary carcinomas are indeed polyclonal. However, there already exists a substantial body of evidence from cytogenetic studies of breast cancers indicating that many of them are polyclonal. Although there is still room for interpretation and some doubt remains as to exactly which role should be ascribed to the observed clonal heterogeneity in our models of carcinogenesis, it seems obvious that more attention than before ought to be paid to this now well documented fact.

Breast Neoplasms↗

Prostate cancer metastasis: role of the host microenvironment in promoting epithelial to mesenchymal transition and increased bone and adrenal gland metastasis.

BACKGROUND: The ARCaP cell line was established from the ascites fluid of a patient with metastatic prostate cancer. This study characterized the host microenvironmental role in cancer progression, epithelial to mesenchymal transition (EMT), and bone and adrenal metastasis in parental ARCaP and its derived cell subclones. METHODS: Cytogenetic profiles, growth, migration, invasion, cellular interaction, drug sensitivities, and gene expression of ARCaP cell subclones were compared. In vivo gene expression, behavior, and metastasis of ARCaP subclones were analyzed by serial intracardiac injections into SCID mice. RESULTS: ARCaP(E) cells, with cobblestone morphology, underwent EMT through cellular interaction with host bone and adrenal gland. Lineage-derived ARCaP(M) cells, with spindle-shape fibroblastic morphology, exhibited decreased cell adhesion and increased metastasis to bone and adrenal gland. Cytogenetic analyses of parental and ARCaP subclones confirmed their clonality. CONCLUSIONS: ARCaP uniquely models the molecular basis of prostate cancer bone and adrenal metastases and epithelial to mesenchymal transition.

Adenocarcinoma↗

Establishment of a human fibroblast cell line producing tumor necrosis factor alpha (KMST-6/TNF) and growth inhibitory effects of its conditioned medium on malignant cells in culture.

To develop a new gene therapy model for cancer, a clonal cell line (KMST-6/TNF) which produces human tumor necrosis factor alpha (hTNF-alpha) has been developed by introducing hTNF-alpha cDNA into a human immortal fibroblast cell line (KMST-6). The conditioned medium (CM) of KMST-6/TNF cells inhibited the growth of various malignant human cell lines, but not that of normal human fibroblasts. Although the growth inhibitory effects of KMST-6/TNF CM were neutralized to a considerable degree by anti-TNF-alpha antibody, its inhibitory effects were more marked than the purified human natural TNF-alpha itself in the same units, suggesting that KMST-6/TNF CM contains some growth inhibitory substances other than TNF-alpha. However, interferons alpha, beta, and gamma were undetectable in the KMST-6/TNF CM.

Blotting, Northern↗

Heme deficiency suppresses the expression of key neuronal genes and causes neuronal cell death.

Defective heme synthesis may cause acute porphyrias, which are associated with a wide array of neurological disturbances involving both the central and peripheral nervous systems. Thus, the understanding of the roles of heme in neuronal cell function may provide insights into the molecular events underlying the pathogenesis of neuropathies associated with defective heme synthesis. In this report, we use rat pheochromocytoma (PC12) clonal cells as a model system for studying the role of heme in neuronal cell survival. We examined the effects of inhibition of heme synthesis on signaling pathways and gene expression in nerve growth factor (NGF)-induced PC12 cells. We found that succinyl acetone-induced heme deficiency selectively caused apoptosis in NGF-induced PC12 cells. Further, we found that in succinyl acetone-treated, NGF-induced cells, the pro-survival Ras-ERK1/2 signaling pathway was inactivated and the pro-apoptotic JNK signaling pathway was activated. In these cells, the activation of caspase and the cleavage of nuclear poly (ADP-ribose) polymerase (PARP) were also evident. Importantly, microarray gene expression analysis showed that more than 20 key neuronal genes that were induced by NGF were suppressed by succinyl acetone. These genes include those encoding survival motor neuron protein, synaptic vesicle protein SVOP, and neural cell adhesion molecule NCAM. These results indicate that heme is important for neuronal cell signaling and the proper functioning of neuronal cells.

Animals↗

Does Cryptosporidium parvum have a clonal population structure?

Cryptosporidiosis, the disease caused in humans by the opportunistic parasite Cryptosporidium parvum, is the result of zoonotic or anthroponotic transmission. Molecular characterization of different isolates from humans and other mammalian species has recently shown this species to be heterogeneous; this heterogeneity has been linked to the host of isolation, suggesting that the parasites causing zoonotic cryptosporidiosis and those propagated by anthroponotic transmission are genetically distinct. Here, Fatih Awad-El-Kariem provides an update on the taxonomic and epidemiological significance of these observations, and discusses evidence for and against the clonality hypothesis as a model to explain strain variation in this species.

Animals↗

Clonal analysis of a bladder cancer cell line: an experimental model of tumour heterogeneity.

The continuous cell line UCRU BL 17CL was derived from a human invasive bladder cancer and expresses elements of transitional, squamous and glandular differentiation. Nine clones of this line were established by limit dilution and have been extensively characterised. Only six of these clones grew subcutaneously in nude mice. Of these, three have exhibited local invasion, each in one of five implanted mice. Although all xenografts expressed transitional, squamous and glandular elements, different histological subtypes predominated within each clone. Only clones which grew in nude mice formed colonies in semi-solid medium, and each responded differently to the influence of medium that had been conditioned by the growth of UCRU BL 17CL, suggesting the possible secretion of a growth factor by these cells. The DNA content and lectin binding profiles of the clones also reflected the heterogeneity of the line. UCRU BL 17CL and the nine clones provide a unique model for the study of tumour heterogeneity, progression and differentiation, and the potential autocrine regulation of growth of bladder cancer.

Carcinoma, Transitional Cell↗

Negative selection--clearing out the bad apples from the T-cell repertoire.

Dead cells are a prominent feature of the thymic landscape as only 5% of developing thymocytes are exported as mature T cells. The remaining thymocytes die by one of two mechanisms; most thymocytes die because they are not positively selected and do not receive a survival signal, whereas a minority of thymocytes undergo T-cell receptor (TCR)-mediated apoptosis, a process known as negative selection. Negative selection is extremely important for establishing a functional immune system, as it provides an efficient mechanism for ridding the T-cell repertoire of self-reactive and potentially autoimmune lymphocytes. This review discusses several cellular and molecular aspects of negative selection.

Apoptosis↗

Selection of rat hepatoma cells defective in hormone-regulated production of mouse mammary tumor virus RNA.

We have been studying the mechanism of glucocorticoid hormone action by using mouse mammary tumor virus (MMTV)-infected rat hepatoma cells as a model system. J2.17, a clonal cell line that contains one MMTV provirus, induces tyrosine aminotransferase (TyrATase; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5), viral RNA, and the cell surface viral glycoprotein gp52 in response to dexamethasone. Using a fluorescence-activated cell sorter and a rabbit antiserum directed against gp52, we selected a cell population that displays a reduced hormone-mediated increase in cell surface gp52. Fourteen clones of this population were assayed for induction of viral gp52 and RNA and of cellular TyrATase. The results of these assays revealed that the clones display a variety of responses to hormone. One clone has retained wild-type responses of both TyrATase and gp52. Six clones exhibit coordinately reduced or abolished responses of both markers. Seven clones show normal induction of TyrATase but reduced or undetectable induction of gp52. These latter clones exhibit reduced production of MMTV RNA and thus may represent a unique class of variants defective in the regulation of MMTV gene expression.

Animals↗

Evidence for the maintenance of hematopoiesis in a large animal by the sequential activation of stem-cell clones.

To test if hematopoiesis can be maintained by the sequential activation of stem-cell clones, we performed autologous marrow transplantations with limited numbers of cells in cats heterozygous for the X chromosome-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) and observed the G6PD phenotypes of erythroid and granulocyte/macrophage progenitors over time. The animals were the female offspring of Geoffroy male and domestic female cats. In repeated studies of marrow from control animals (n = 5) or experimental animals prior to transplantation (n = 3), the percent of progenitors with domestic-type G6PD did not vary. After transplantation, the peripheral blood counts, marrow morphologies, frequencies of progenitors, and progenitor cell cycle kinetics returned to normal. However, abrupt and significant fluctuations were seen in the G6PD type of progenitors from each cat during the 1-1.5 years of observation. These data cannot be explained if there were either a large or constant population of active stem cells and thus imply, in a large-animal system, that hematopoiesis was maintained through clonal succession. A stochastic model was developed to estimate the numbers of active clones and their mean lifetimes.

Animals↗

Gene expression profiling of initiated epidermal cells with benign or malignant tumor fates.

High-density oligonucleotide array technology was used to search for gene expression changes that may predict or cause sporadic tumorigenesis. To reduce variation among samples, a non-transformed mouse epidermal keratinocyte clone was compared with its carcinoma-producing or papilloma-producing 7,12-dimethylbenz[a]anthracene-initiated cell derivative. The majority of the approximately 12 325 genes or expressed sequence tags (ESTs) analyzed remained unaltered reflective of the clonal nature of the model. Consistent gene expression changes among biological replicates included 69 in malignancy-prone initiated cells, 46 in papilloma-precursor initiated cells and an additional 45 with changes in both initiated cell lineages. The changes covered a broad spectrum of cellular activities, implying that multiple pathways cooperate at the initiation of carcinogenesis. There was a tendency in the malignancy-prone cells for differences in proliferation and apoptosis-related genes. The benign lineage tended toward aberrant expression of genes involved in differentiation and epidermal barrier function. Several independently confirmed gene expression changes revealed plausible effectors to bypass an activated ras protein in proliferation pathways and to compromise the wild-type p53 and other apoptosis pathways at initiation of carcinogenesis. The differential expression patterns of the initiated cells are consistent with the hypothesis that changes in gene expression at initiation may cooperate with subsequent oncogenic changes to determine malignant fate. The cloned epidermal cell lineages allowed detection of putative early cancer genes in a model that is conducive to testing their direct role in epithelial multistep carcinogenesis in vitro and in vivo.

Animals↗

Differences in response to a chemical carcinogen within species and clones of the livebearing fish, Poeciliopsis.

Freshwater viviparous fishes of the genus Poeciliopsis are evaluated as a model for testing water-borne carcinogens. The unique property of this model lies in the clonal form of reproduction of some of these fishes. Interspecific hybrids between P.monacha and P.lucida occur in nature as all female 'species' which produce only female offspring and transmit the P.monacha genome intact as a single linkage group. Without sacrificing the integrity of the wild P.monacha genome, a naturally occurring 'hemi-clone' can be brought into the laboratory and rendered isogenic in the next generation by providing an inbred strain of P.lucida as the male parent. The genetic mechanism of P.monacha-lucida hybrids provides a tool for duplicating wild genomes from heterogeneous populations of these fishes for broad and continuous investigation. In the present study susceptibility to induction of liver tumors is compared in inbred strains of two species Poeciliopsis lucida and P.monacha, and among nine P.monacha-lucida hybrid clones exposed to diethylnitrosamine (DEN). Incidence of fish with liver tumors was significantly higher in P.lucida M61-31 (89.0%) than in P.monacha strains S68-4 (18.9%) and S68-5 (36.8%) and the nine hybrid clones of P.monacha-lucida wherein tumors occurred in a graded series (15.6 to 66.7%). Tumors were not found in any untreated fish reared as controls. The incidence of treated fish with large tumors (occupying more than one eighth of the liver) was significantly higher in P.lucida (72.7%) than in the P.monacha strains S68-4 (9.4%) and S68-5 (10.5%). The difference in frequencies of large tumors among the nine clones ranges from a low of 3.8% in M65-24 PCx to a high of 52.9% in M61-35 PCz. Hemangiopericytomas were induced in P.lucida and in five of the nine P.monacha-lucida clones; they were not found in either strain of P.monacha. Lymphosarcoma was induced only in P.monacha S68-5. An unclassified sarcoma was induced in both P.monacha strains and in two of the nine P.monacha-lucida clones, but not in P.lucida. The intrahepatic cholangiocarcinoma was the most frequent tumor type induced with DEN in this study. It occurred in all twelve genotypes, and with one exception, in the highest incidence. In contrast to a previous study wherein responses of different strains of P.lucida were within a narrow range, substantial differences exist among P.monacha genotypes.

Animals↗

The effect of autocorrelation in environmental variability on the persistence of populations: an experimental test.

Despite its significance regarding the conservation and management of biological resources, the body of theory predicting that the correlation between successive environmental states can profoundly influence extinction has not been empirically validated. Identical clonal populations from a model experimental system based on the collembolan Folsomia candida were used in the present study to investigate the effect of environmental autocorrelation on time to extinction. Environmental variation was imposed by variable implementation (present/absent) of a culling procedure according to treatments that represented six patterns of environmental autocorrelation. The average number of culling events was held constant across treatments but, as environmental autocorrelation increased, longer runs of both favourable and unfavourable culling tended to occur. While no difference was found among the survival functions for the various treatments, the time taken for 50% of the component populations to become extinct decreased significantly with increasing environmental autocorrelation. Similarly, analysis of all extinct populations demonstrated that time to extinction was shortened as environmental autocorrelation increased. However, this acceleration of extinction can be fully offset if sequential introduction is used in place of simultaneous introduction when founding the populations.

Animals↗

Host MHC class I gene control of NK-cell specificity in the mouse.

The missing self model predicts that NK cells adapt somatically to the type as well as levels of MHC class I products expressed by their host. Transgenic and gene knock-out mice have provided conclusive evidence that MHC class I genes control specificity and tolerance of NK cells. The article describes this control and discusses the possible mechanisms behind it, starting from a genetic model to study how natural resistance to tumors is influenced by MHC class I expression in the host as well as in the target cells. Data on host gene regulation of NK-cell functional specificity as well as Ly49 receptor expression are reviewed, leading up to the central question: how does the system develop and maintain "useful" NK cells, while avoiding "harmful" and "useless" ones? The available data can be fitted within each of two mutually none-exclusive models: cellular adaptation and clonal selection. Recent studies supporting cellular adaptation bring the focus on different possibilities within this general mechanism, such as anergy, receptor calibration and, most importantly, whether the specificity of each NK cell is permanently fixed or subject to continuous regulation.

Animals↗

Surface expression of two distinct functional antigen receptors on human gamma delta T cells.

Lymphocytes recognize antigens with highly variable heterodimeric surface receptors. Although four distinct antigen receptors could in principle be produced by any lymphocyte, only one functional combination of receptor chains has thus far been found expressed on their surface. Examination of human gamma delta T cells revealed a population that violated this rule by expressing on their surface two distinct functional gamma delta T cell receptors (TCRs) that used different TCR gamma gene alleles. Thus, current models for T cell clonal selection may need modification, and a possible escape mechanism for autoreactive TCRs is suggested.

Alleles↗

Bacillus anthracis virulence in Guinea pigs vaccinated with anthrax vaccine adsorbed is linked to plasmid quantities and clonality.

Bacillus anthracis is a bacterial pathogen of great importance, both historically and in the present. This study presents data collected from several investigations and indicates that B. anthracis virulence is associated with the clonality and virulence of plasmids pXO1 and pXO2. Guinea pigs vaccinated with Anthrax Vaccine Adsorbed were challenged with 20 B. anthracis isolates representative of worldwide genetic diversity. These same isolates were characterized with respect to plasmid copy number by using a novel method of quantitative PCR developed for rapid and efficient detection of B. anthracis from environmental samples. We found that the copy numbers for both pXO1 and pXO2 differed from those in previously published reports. By combining the data on survival, plasmid copy numbers, and clonality, we developed a model predicting virulence. This model was validated by using a randomly chosen set of 12 additional B. anthracis isolates. Results from this study will be helpful in future efforts to elucidate the basis for variation in the virulence of this important pathogen.

Animals↗

Characterization of the T-cell repertoire in autologous graft-versus-host disease (GVHD): evidence for the involvement of antigen-driven T-cell response in the development of autologous GVHD.

Administration of cyclosporine A (CsA) after autologous stem cell transplantation elicits an autoimmune syndrome with pathology similar to graft-versus-host disease (GVHD). This syndrome, termed autologous GVHD, is associated with the appearance of autoreactive T cells directed at major histocompatibility class (MHC) class II antigens. In the rat model of autologous GVHD, clonal analysis reveals that the effector T cells are highly conserved and recognize a peptide from the invariant chain peptide presented by MHC class II. Although human autologous GVHD effector T cells share a similar phenotypic specificity, clonality of the response in humans has not been determined. To examine the human effector T-cell response, the T-cell repertoire of peripheral blood lymphocytes was assessed by complementarity-determining region 3 (CDR3) size distribution analysis and T-cell clonotype analysis in 26 patients treated with CsA after transplantation. Autologous GVHD developed in 3 of 4 patients with human leukocyte antigen (HLA)-DRB1*0701, and clonal expansions of beta-chain variable region (BV)16(+) T cells were shared. Clonal expansions within BV15(+) and BV22(+) T cells were also detected in 4 of 6 patients with HLA-DRB1*1501 and in 3 of 4 patients with HLA-DRB1*0401, respectively. Sequencing of BV16 cDNA for which the CDR3 size pattern exhibited apparent clone predominance revealed an identical CDR3 peptide sequence in 2 different patients, one with HLA-DRB1*0701 and the other with HLA-DRB1*1502. These findings indicate that the discrete antigen-driven expansion of T cells is involved in autologous GVHD. (Blood. 2001;98:868-876)

Adult↗

T cell immunity induced by live, necrotic, and apoptotic tumor cells.

The rules that govern the engagement of antitumor immunity are not yet fully understood. Ags expressed by tumor cells are prone to induce T cell tolerance unless the innate immune system is activated. It is unclear to what extent tumors engage this second signal link by the innate immune system. Apoptotic and necrotic (tumor) cells are readily recognized and phagocytosed by the cells of the innate immune system. It is unknown how this affects the tumor's immunogenicity. Using a murine melanoma (B16m) and lymphoma (L5178Y-R) model, we studied the clonal sizes and cytokine signatures of the T cells induced by these tumors in syngeneic mice when injected as live, apoptotic, and necrotic cells. Both live tumors induced a type 2 CD4 cell response characterized by the prevalent production of IL-2, IL-4, and IL-5 over IFN-gamma. Live, apoptotic, and necrotic cells induced CD4 (but no CD8) T cells of comparable frequencies and cytokine profiles. Therefore, live tumors engaged the second signal link, and apoptotic or necrotic tumor cell death did not change the magnitude or quality of the antitumor response. A subclone of L5178Y-R, L5178Y-S cells, were found to induce a high-frequency type 1 response by CD4 and CD8 cells that conveyed immune protection. The data suggest that the immunogenicity of tumors, and their characteristics to induce type 1 or type 2, CD4 or CD8 cell immunity is not primarily governed by signals associated with apoptotic or necrotic cell death, but is an intrinsic feature of the tumor itself.

Animals↗