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PubMed2002

Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia.

Through sequencing analysis of blood or bone marrow samples from patients with chronic myeloid leukemia, we identified BCR-ABL kinase domain mutations in 29 of 32 patients whose disease relapsed after an initial response to the tyrosine kinase inhibitor imatinib. Fifteen different amino acid substitutions affecting 13 residues in the kinase domain were found. Mutations fell into two groups-those that alter amino acids that directly contact imatinib and those postulated to prevent BCR-ABL from achieving the inactive conformational state required for imatinib binding. Distinct mutations conferred varying degrees of imatinib resistance. Mutations detected in a subset of patients with stable chronic phase disease correlated with subsequent disease progression. Multiple independent mutant clones were detected in a subset of relapsed cases. Our data support a clonal selection model of preexisting BCR-ABL mutations that confer imatinib resistance.

Alleles↗

PubMed2005

The role of pancreatic cyst fluid molecular analysis in predicting cyst pathology.

BACKGROUND & AIMS: Current methods to detect malignancy in mucinous cystic neoplasms of the pancreas remain inadequate. The role of detailed molecular analysis in this context was investigated. METHODS: Endoscopic ultrasound-guided pancreatic cyst aspirates were prospectively collected during a period of 19 months and studied for cytology, carcinoembryonic antigen level, and molecular analysis. Molecular evaluation incorporated DNA quantification (amount and quality), k-ras point mutation, and broad panel tumor suppressor linked microsatellite marker allelic loss analysis by using fluorescent capillary electrophoresis. The sequence of mutation acquisition was also calculated on the basis of a clonal expansion model, and comparison was made to the final pathology. RESULTS: Thirty-six cysts with confirmed histology were analyzed. There were 11 malignant, 15 premalignant, and 10 benign cysts. Malignant cysts could be differentiated from premalignant cysts on the basis of fluid carcinoembryonic antigen level (P=.034), DNA quality (P=.009), number of mutations (P=.002), and on the sequence of mutations acquired (P<.001). Early k-ras mutation followed by allelic loss was the most predictive of a malignant cyst (sensitivity, 91%; specificity, 93%). CONCLUSIONS: Malignant cyst fluid contains adequate DNA to allow mutational analysis. A first hit k-ras mutation followed by allelic loss is most predictive of the presence of malignancy in a pancreatic cyst. This approach should serve as an ancillary tool to the conventional work-up of pancreatic cysts. Cumulative amount and timing of detectable mutational damage can assist in diagnosis and clinical management.

Carcinoembryonic Antigen↗

PubMed2002

Chagas disease: role of parasite genetic variation in pathogenesis.

Chagas disease, caused by the parasite protozoan Trypanosoma cruzi, is characterised by a variable clinical course, from symptomless cases to severe chronic disease with cardiac and/or gastrointestinal involvement. This variability has been attributed both to differences in the host response and to genomic heterogeneity of the parasite. This article reviews the evidence in favour of an important role of the genetic constitution of T. cruzi in determining the clinical characteristics of Chagas disease and discusses the basis of the 'Clonal-Histotropic Model' for the pathogenesis of this disease.

Animals↗

PubMed2001

A comparative analysis of the immunological evolution of antibody 28B4.

In an effort to gain greater insight into the evolution of the redox active, catalytic antibody 28B4, the germline genes used by the mouse to generate this antibody were cloned and expressed, and the X-ray crystal structures of the unliganded and hapten-bound germline Fab of antibody 28B4 were determined. Comparison with the previously determined structures of the unliganded and hapten-bound affinity-matured Fab [Hsieh-Wilson, L. C., Schultz, P. G., and Stevens, R. C. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 5363] shows that the germline antibody binds the p-nitrophenyl ring of hapten 3 in an orientation significantly different from that seen in the affinity-matured antibody, whereas the phosphonate moiety is bound in a similar mode by both antibodies. The affinity-matured antibody 28B4 has more electrostatic and hydrophobic interactions with hapten 3 than the germline antibody and binds the hapten in a lock-and-key fashion. In contrast, significant conformational changes occur in the loops of CDR H3 and CDR L1 upon hapten binding to the germline antibody, consistent with the notion of structural plasticity in the germline antibody-combining site [Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G., and Stevens, R. C. (1997) Science 276, 1665]. The structural differences are reflected in the differential binding affinities of the germline Fab (K(d) = 25 microM) and 28B4 Fab (K(d) = 37 nM) to hapten 3. Nine replacement mutations were found to accumulate in the affinity-matured antibody 28B4 compared to its germline precursor. The effects of each mutation on the binding affinity of the antibody to hapten 3 were characterized in detail in the contexts of both the germline and the affinity-matured antibodies. One of the mutations, Asp95(H)Trp, leads to a change in the orientation of the bound hapten, and its presence is a prerequisite for other somatic mutations to enhance the binding affinity of the germline antibody for hapten 3. Thus, the germline antibody of 28B4 acquired functionally important mutations in a stepwise manner, which fits into a multicycle mutation, affinity selection, and clonal expansion model for germline antibody evolution. Two other antibodies, 20-1 and NZA6, with very different antigen specificities were found to be highly homologous to the germline antibody of 28B4, consistent with the notion that certain germline variable-region gene combinations can give rise to polyspecific hapten binding sites [Romesberg, F. E., Spiller, B., Schultz, P. G., and Stevens, R. C. (1998) Science 279, 1929]. The ultimate specificity of the polyspecific germline antibody appears to be defined by CDR H3 variability and subsequent somatic mutation. Insights into the evolution of antibody-combining sites provided by this and other structural studies are discussed.

Amino Acid Sequence↗

PubMed1998

Estimation of unit risk for coke oven emissions.

In 1984, based on epidemiological data on cohorts of coke oven workers, USEPA estimated a unit risk for lung cancer associated with continuous exposure from birth to 1 microgram/m3 of coke oven emissions, of 6.2 x 10(-4). This risk assessment was based on information on the cohorts available through 1966. Follow-up of these cohorts has now been extended to 1982 and, moreover, individual job histories, which were not available in 1984, have been constructed. In this study, lung cancer mortality in these cohorts of coke oven workers with extended follow-up was analyzed using standard techniques of survival analysis and a new approach based on the two stage clonal expansion model of carcinogenesis. The latter approach allows the explicit consideration of detailed patterns of exposure of each individual in the cohort. The analyses used the extended follow-up data through 1982 and the detailed job histories now available. Based on these analyses, the best estimate of unit risk is 1.5 x 10(-4) with 95% confidence interval = 1.2 x 10(-4)-1.8 x 10(-4).

Aged↗

PubMed1980

Tolerance as an active process.

The clonal deletion model proposed by Burnet and Lederberg and expanded by Nossal was one of the first theories concerning the nature of tolerance of self constituents. The model proposed that during the maturation of lymphocytes into immunocompetent cells, there is a sensitive differentiation stage whereby contact wth antigen results in specific inactivation of the cell. Experimental evidence indicates that neonatal or immature B lymphocytes are indeed different from adult lymphocytes in their extreme sensitivity to tolerance induction even at low antigen concentrations and with antigens that are normally immunogenic. The present study examines the mechanism of this tolerance phenomenon by determining whether or not tolerance of immature B cells is an active process and what specific interactions can induce this event. We used various putative inhibitors of tolerance induction in the splenic focus assay which examines the tolerance susceptibility of individual B cells. The results suggest that tolerance requires protein synthesis and that this process is initiated only after a minimum threshold affinity of binding occurs between antigen and cell-surface receptor with subsequent receptor interlinkage.

Animals↗

PubMed1992

Clonal expansion of p53 mutant cells is associated with brain tumour progression.

Tumour progression is a fundamental feature of the biology of cancer. Cancers do not arise de novo in their final form, but begin as small, indolent growths, which gradually acquire characteristics associated with malignancy. In the brain, for example, low-grade tumours (astrocytomas) evolve into faster growing, more dysplastic and invasive high-grade tumours (glioblastomas). To define the genetic events underlying brain tumour progression, we analysed the p53 gene in ten primary brain tumour pairs. Seven pairs consisted of tumours that were high grade both at presentation and recurrence (group A) and three pairs consisted of low-grade tumours that had progressed to higher grade tumours (group B). In group A pairs, four of the recurrent tumours contained a p53 gene mutation; in three of them, the same mutation was found in the primary tumour. In group B pairs, progression to high grade was associated with a p53 gene mutation. A subpopulation of cells were present in the low-grade tumours that contained the same p53 gene mutation predominant in the cells of the recurrent tumours that had progressed to glioblastoma. Thus, the histological progression of brain tumours was associated with a clonal expansion of cells that had previously acquired a mutation in the p53 gene, endowing them with a selective growth advantage. These experimental observations strongly support Nowell's clonal evolution model of tumour progression.

Amino Acid Sequence↗

PubMed1995

Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas.

A number of murine T-cell hybridomas undergo apoptosis within a few hours of activation by specific antigens, mitogens, antibodies against the T-cell antigen receptor, or a combination of phorbol ester and calcium ionophore. This phenomenon has been extensively studied as a model for clonal deletion in the immune system, in which potentially autoreactive T cells eliminate themselves by apoptosis after activation, either in the thymus or in the periphery. Here we show that the Fas/CD95 receptor, which can transduce a potent apoptotic signal when ligand, is rapidly expressed following activation of T-cell hybridomas, as is its functional, membrane-bound ligand. Interference with the ensuing Fas/Fas-ligand interaction inhibits activation-induced apoptosis. Because T-cell receptor ligation can induce apoptosis in a single T hybridoma cell, we suggest that the Fas/Fas-ligand interaction can induce cell death in a cell-autonomous manner.

Animals↗

PubMed2006

Individual stem cells with highly variable proliferation and self-renewal properties comprise the human hematopoietic stem cell compartment.

Hematopoiesis requires tight regulation of the hematopoietic stem cell (HSC) population; however, the dynamics of HSC use at steady state are uncertain. Over 3-7 months, we evaluated the repopulation and self-renewal of more than 600 individual human 'severe combined immunodeficiency mouse-repopulating cells' (SRCs), tracked on the basis of lentiviral integration sites, in serially transplanted immune-deficient mice, as well as of SRC daughter cells that migrated to different marrow locations in a single mouse. Our data demonstrate maintenance by self-renewing SRCs after an initial period of clonal instability, a result inconsistent with the clonal succession model. We found wide variation in proliferation kinetics and self-renewal among SRCs, as well as between SRC daughter cells that repopulated equivalently, suggesting that SRC fate is unpredictable before SRCs enter more rigid 'downstream' developmental programs.

ADP-ribosyl Cyclase 1↗

PubMed1999

Cells with TP53 mutations in low grade astrocytic tumors evolve clonally to malignancy and are an unfavorable prognostic factor.

It is important to understand how low grade tumors recur and progress to malignant lesions since this dramatically shortens patient survival. Here, we evaluated the concept that malignant progression and poor prognosis of low grade astrocytic tumors are TP53 dependent through clonal expansion of mutated cells. TP53 status was established in primary and recurrent tumors from 36 patients with WHO grade II astrocytic tumors and two tumor types were found. Tumors from 14 patients (39%; type 1) had TP53 mutated cells, and 92% of these recurred with 57% progressing to malignancy. The evolution of TP53 mutated cells before and after progression was examined using a clonal analysis procedure in yeast. Malignant progression was accompanied by an increased percentage of mutant TP53 (red) yeast colonies resulting from monoclonal expansion of cells with mutated TP53. The presence of TP53 mutations in WHO grade II astrocytic tumors was associated with malignant progression (P=0. 034, chi2 test) and shorter progression-free survival (PFS; 47.6+/-9. 6 months for TP53-mutated tumors vs 67.8+/-8.2 months for TP53-wild type tumors, P<0.05, log-rank test). Tumors from 22 patients (61%; type 2) were without TP53 mutations, and 64% of these recurred without a change in TP53 status, although 41% progressed to malignancy. This suggests that TP53 mutation is not an initiating or progression event in the majority of low grade astrocytic tumors. Our study also indicates that irradiation for WHO grade II astrocytic tumors might be associated with poor outcome (P<0.0001) and this was independent of TP53 status. These findings have important implications in the clinical management of patients with low grade astocytoma and provide new support to the clonal evolution model for tumor progression.

Adolescent↗

PubMed2000

SPAF, a new AAA-protein specific to early spermatogenesis and malignant conversion.

A novel spermatogenesis associated factor (SPAF) was found to be aberrantly expressed at the malignant conversion stage in a clonal epidermal model of chemical carcinogenesis. Sequence analysis revealed two ATPase modules, classifying this gene as a new member of the AAA-protein family (ATPase associated with diverse activities). Immunohistochemical staining of mouse testis sections with SPAF antibody localized expression to spermatogonia and early spermatocytes in the basal compartment of the seminiferous tubules. Northern and Western analysis of SPAF expression in testes of mice at different developmental stages confirmed its expression at early stages of spermatogenesis. In view of a mitochondrial-localization-like signal, sequence similarities to membrane-associated proteins, ATP binding properties, and intracellular expression patterns in testis, we speculate that SPAF protein may be involved in morphological and functional mitochondrial transformations during spermatogenesis. Ectopic expression of the SPAF gene in malignant epidermal cells may signify adoption of an early germ cell-like phenotype advantageous in malignant conversion.

Adenosine Triphosphatases↗

PubMed2000

Retinoic acid-mediated enhancement of the cholinergic/neuronal nitric oxide synthase phenotype of the medial septal SN56 clone: establishment of a nitric oxide-sensitive proapoptotic state.

It is unclear what mechanisms lead to the degeneration of basal forebrain cholinergic neurons in Alzheimer's or other human brain diseases. Some brain cholinergic neurons express neuronal nitric oxide (NO) synthase (nNOS), which produces a free radical that has been implicated in some forms of neurodegeneration. We investigated nNOS expression and NO toxicity in SN56 cells, a clonal cholinergic model derived from the medial septum of the mouse basal forebrain. We show here that, in addition to expressing choline acetyltransferase (ChAT), SN56 cells express nNOS. Treatment of SN56 cells with retinoic acid (RA; 1 microM) for 48 h increased ChAT mRNA (+126%), protein (+88%), and activity (+215%) and increased nNOS mRNA (+98%), protein (+400%), and activity (+15%). After RA treatment, SN56 cells became vulnerable to NO excess generated with S-nitro-N-acetyl-DL-penicillamine (SNAP) and exhibited increased nuclear DNA fragmentation that was blocked with a caspase-3 inhibitor. Treatment with dexamethasone, which largely blocked the RA-mediated increase in nNOS expression, or inhibition of nNOS activity with methylthiocitrulline strongly potentiated the apoptotic response to SNAP in RA-treated SN56 cells. Caspase-3 activity was reduced when SNAP was incubated with cells or cell lysates, suggesting that NO can directly inhibit the protease. Thus, whereas RA treatment converts SN56 cells to a proapoptotic state sensitive to NO excess, endogenously produced NO appears to be anti-apoptotic, possibly by tonically inhibiting caspase-3.

Acetylcholine↗

PubMed1986

Clonal expansion of T cells: a cytotoxic T-cell response in vivo that involves precursor cell proliferation.

The response of peritoneal exudate lymphocytes to allogeneic tumor cells was used to determine whether the in vivo generation of cytotoxic T cells (CTL) involved the proliferation of precursor cells. Ten days post-injection, both cytotoxic activity and the formation of conjugates between lymphocytes and target cells were shown to be specific for the immunizing tumor alloantigens and to be effected by Ly-2+ cells. A cell-sorting-based procedure was developed to isolate specific conjugates between red-fluorescence-tagged CTL and blue-fluorescence-tagged tumor target cells. When [3H]thymidine was administered during the response, almost all isolated conjugate-forming CTL were 3H-labeled on autoradiography. Thus, the CTL were clearly products of dividing cells, a result that contradicts published data. Reassessment of a previously studied system, which suggested that CTL were not products of cell division, indicated that in that system many of the conjugate-forming cytotoxic cells studied were Ly-2- and nonspecific, and thus perhaps not T cells. We conclude that the clonal selection model is applicable to at least one in vivo T-cell response.

Animals↗

PubMed1988

Number and continuous proliferative pattern of transplanted primitive immunohematopoietic stem cells.

We estimated numbers of transplantable primitive stem cells (PSCs) and found evidence that the same PSC continuously produced circulating erythrocytes and lymphocytes. These estimations used the binomial formula on data from recipients of identical portions of marrow mixtures containing two distinguishable cell types. Analysis of variance was used to compare repeated tests within each recipient. Values of pi s or pi c, probabilities that two independently sampled cells were descended from the same PSC, were also estimated, as this does not require the unverified condition that all PSCs contribute equally to the differentiated cell population. Several months after transplantation, erythrocytes were descended from only a single PSC per 1-2 X 10(5) marrow cells injected, several times rarer than previously reported. Percentages of erythrocyte and lymphocyte types in each recipient were closely correlated, with r values ranging from 0.86 to 0.94, in groups receiving 2-8 X 10(5) marrow cells; apparently the same precursors repopulated both myeloid and lymphoid lines in each recipient, as expected of true PSCs. Our data did not fit the clonal succession model, which predicts sequential activation of new PSCs and deactivation of old. Between 76 and 154 days, differentiated erythrocyte precursors were probably exhausted, with no evidence for new precursor activation or for further change between 154 and 250 days. The percentage of newly produced erythrocytes (reticulocytes) of each donor type varied little when individual recipients were followed between 165 and 295 days after transplantation, and variances within recipients were similar at marrow doses from 8 to 200 X 10(5) cells, further contradicting models of sequential activation and deactivation of PSC clones. Thus, transplanted PSCs were continually active during much of the recipient's lifespan.

Analysis of Variance↗

PubMed2000

Molecular and environmental causes of cancer.

The multistep, clonal evolution model of cancer predicts fundamental mechanisms by which chemicals may influence the cancer process. These are illustrated by studies of the mechanisms of induction of carcinogenesis by estrogens and the inhibition of carcinogenesis by caloric restriction.

Carcinogens, Environmental↗

PubMed1982

Generation of T cell colonies from responder strain 2 guinea pigs that recognize the copolymer L-glutamic acid, L-lysine in association with nonresponder strain 13 Ia antigens.

T cells from nonimmune responder strain 2 guinea pigs were primed in vitro to the copolymer GL in association with allogeneic, nonresponder strain 13 PEC. T cells that recognized GL in association with strain 13 Ia were separated from alloreactive T cells by cloning the population in soft agar following the priming in liquid culture. The existence of T cells of responder origin that recognize antigen in association with nonresponder macrophages is most consistent with clonal deletion models of Ir gene function.

Animals↗

PubMed2002

Signals for a promoting action of radiation in cancer incidence data.

The two-stage clonal expansion model allows us in principle to separate the initiating and promoting action of radiation. Features of the relative risk functions which indicate the action of promotion most clearly are identified for acute and for protracted exposures. They are compared with data: for acute exposure, some results of a preliminary analysis of the atomic-bomb survivor data are given; for protracted exposure, patterns in the fatal lung tumours of radon-exposed rats are discussed.

Animals↗

PubMed2005

Identification of the starting point for spermatogenesis and characterization of the testicular stem cell in adult male rhesus monkeys.

BACKGROUND: Spermatogonial expansion in man and non-human primates has been studied for decades. Controversy persists about the cell type representing the testicular stem cell and the exact kinetics of spermatogonial proliferation. We recently determined the starting point of spermatogenesis and proposed a model for clonal expansion of spermatogonia in adult macaques. Here we want to confirm the initiation event, study and compare the details of the kinetics of spermatogonial expansion in vivo and in vitro, and characterize a population of A spermatogonia acting as testicular stem cells. METHODS AND RESULTS: We localized BrdU-positive spermatogonia in whole mounts and sections of adult rhesus monkey testes. Culture of testicular tissue was used to determine the expansion and differentiation of premeiotic germ cells. We confirm that A(pale) spermatogonia divide equally at stage VII and produce two types of progeny after mitosis at stage IX of the seminiferous cycle following defined clonal patterns. Small numbers of proliferating single A spermatogonia exist which present a population of label-retaining cells. CONCLUSIONS: In the rhesus monkey the population of A(pale) spermatogonia cycle continuously and initiate spermatogenesis by a self-renewing division at stage VII of the seminiferous epithelial cycle. Rarely dividing single A spermatogonia exist which potentially are the male germline stem cells in the primate testis.

Animals↗