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The principal component analysis of 21 chlorella cell survival curves, adjusted by one-hit and two-hit target models, lead to quite similar projections on the principal plan: the homologous parameters of these models are linearly correlated; the reason for the statistical equivalence of these two models, in the present state of experimental inaccuracy, is revealed.
In the clonal human rhabdomyosarcoma cell line TE-671-1A, the expression of genes implicated in myogenic differentiation was determined before and after exposure to the differentiation inducers retinoic acid (RA), sodium butyrate (NaBut) and N-monomethylformamide (NMF). Exposure to NaBut or RA resulted in a significant (NaBut: p < 0.0001; RA: p < 0.05) increase in biochemical differentiation paralleled by a significant (NaBut: p < 0.0001; RA: p < 0.0002) inhibition of proliferation. An increase in the relative number of myotube-like giant cells was observed after exposure to NaBut. Exposure to NMF proved to be least effective and produced a significant (p < 0.0001) inhibition of proliferation without increase in differentiation. On the molecular level, exposure to RA resulted in a moderate increase in RAR a mRNA expression, whereas CRABP mRNA remained constant. RAR beta and RAR gamma mRNA were not expressed. mRNA expression of c-raf, c-myc and c-Ki-ras remained constant before and after exposure to all inducers of differentiation. C-fos mRNA was not expressed. In summary, differentiation can successfully be induced in the human rhabdomyosarcoma cell line TE-671-1A by various inducers of differentiation. In contrast to other myogenic cell lines, however, the proto-oncogenes myc, fos and raf are not involved in the transmission of myogenic differentiation signals in TE-671-1A cells.
A fundamental prediction of the hypothesis that critical events in tumour formation are rare is that the tumours develop by clonal expansion. This prediction has been tested by establishing the isozyme content of tumours induced in mosaic animals whose normal tissues contain two distinguishable isozyme activities. The observation that tumours contain activity associated with one or other of the two cell types present in non-neoplastic tissue has been taken to support the contention that the tumours are clonal in origin. Conversely, the presence of both isozyme activities has been taken to mean that the tumours are derived from a large number of cells. There are conflicting reports concerning the clonal origins of chemically induced epidermal tumours. In this report we present evidence that permits a critical resolution of this controversy. The neoplastic epidermal component of the tumours in mosaic animals was separated from the non-neoplastic dermal components. The neoplastic components were derived exclusively from one or other of the cell lineages present within the mosaic, while the dermal non-neoplastic tissue was shown to be derived from both cell lineages. The data support the view that, irrespective of dose or type of carcinogen, the neoplastic portion of a tumour is clonally derived and that analysis of unseparated tumours is confounded by non-neoplastic dermal tissue. The patch size (number of cells in aggregates of like cell type) was established in the target tissue, isolated epidermis.(ABSTRACT TRUNCATED AT 250 WORDS)
The RT4 neurotumor cell system consists of clonally derived cell lines where a stem cell type segregates in vitro into three biochemically and morphologically different cell types, one glial and two neuronal types. This process has been termed cell-type conversion (M. Imada and N. Sueoka, 1978, Dev. Biol. 66, 97-108). Detailed cytogenetic analysis of the RT4 cell lines are described. Giemsa-banding analysis of 12 independent clonal isolates of the four different RT4 cell types showed a relatively stable karyotype. The stem cell line, RT4-AC, is diploid and most stable, and it has one 4q+ marker chromosome in place of a normal No. 4. This 4q+ marker was identified in all cell types of the RT4 system and was not observed in other cell lines of BDIX origin. The 4q+, therefore, is a chromosomal marker of the RT4 system. Consistent chromosome rearrangement was not found in any one of the cell-type conversions of the RT4-AC cells into the three derivative cell types. The relative stability of the karyotype of the different clonal isolates gives the RT4 system an advantage in studies of genetic regulation and expression of cell-type conversion in vitro. Also the 4q+ marker can be used to identify RT4 cells in coculture experiments or to distinguish RT4 cells in cases of suspected cell-line contamination.
Cutaneous T cell lymphoma (CTCL) and reactive T cell skin diseases represent opposite ends of a spectrum of diseases ranging from overtly malignant to persistently benign. Within this spectrum, the parapsoriasis group is not clearly defined regarding malignant potential. In contrast to consistent findings in advanced-stage CTCL, clonality analysis of parapsoriasis has produced conflicting results in previous studies. As T cell receptor gamma-chain polymerase chain reaction GeneScan analysis (TCR-gamma-PCR-GSA) stands out by its sensitivity, its accuracy in size determination of PCR products, its capacity to identify false positives by repeated analysis and its easy applicability, this approach was used to analyse the clonality status of 41 patients with borderline T cell lymphoproliferative skin diseases, including parapsoriasis (n=27) and early-stage mycosis fungoides (MF) (n=14). A monoclonal T cell infiltrate was demonstrated by repeated TCR-gamma-PCR-GSA in lesional skin specimens in 19.2% of parapsoriasis patients and in 66.6% of early-stage MF cases (p=0.013). In peripheral blood, a monoclonal T cell population was found in a similar percentage of parapsoriasis and of early-stage MF patients (26.7% versus 12.5%; p=0.611). A detailed analysis of parapsoriasis subentities, namely small and large plaque parapsoriasis, and parapsoriasis lichenoides, revealed monoclonality in 2(6)/2(5), 3(14)/2(8) and 0(6)/0/(3) of the skin and peripheral blood specimens, respectively. The high detection rate of false positive cases by repeated analysis (20-37.5%) provides a corrected perspective for the high rates of dominant T cell clones found by others in the peripheral blood of such patients. From the results obtained, three major conclusions can be drawn: firstly, CTCL is clearly associated with detection of monoclonality, even in its early stages; secondly, monoclonality is not a prerequisite for potential CTCL precursor entities; and thirdly, recirculating malignant T cells identical to the skin clone are not readily detected in parapsoriasis or early-stage MF, but may rather indicate disease progression.
Tumor cells represent a single clone of cells that have undergone a series of mutations in genomic DNA. This process, known as clonal evolution, is a distinguishing feature of cancer. The human androgen receptor gene (HUMARA; GenBank) contains a highly polymorphic cytosine-adenine-guanine trinucleotide repeat that can be used to determine clonality by depicting X chromosome inactivation patterns. Random X chromosome inactivation is consistent with polyclonality; nonrandom X chromosome inactivation indicates a clonal population of cells. Basal cell carcinoma (BCC) demonstrates an atypical growth pattern in that it grows slowly, rarely metastasizes, and is rarely lethal. Whether this tumor results from the accumulation of mutations in a single cell with subsequent clonal expansion or reflects a polyclonal response by a group of cells to a growth stimulus is unknown. To provide further insight into the molecular events characterizing BCCs, we determined the clonal origin of five modular BCCs from a female patient by analyzing X chromosome inactivation patterns at the HUMARA locus. All tumors demonstrated a nonrandom pattern of X chromosome inactivation, consistent with monoclonal proliferation. These findings provide strong genetic evidence that sporadic BCCs develop by clonal evolution and support the contention that a series of mutations in a single cell is responsible for the altered growth state seen in these transformed epithelial cells.
The African trypanosome, Trypanosoma brucei, has been shown to undergo genetic exchange in the laboratory, but controversy exists as to the role of genetic exchange in natural populations. Much of the analysis to date has been derived from isoenzyme or randomly amplified polymorphic DNA data with parasite material from a range of hosts and geographical locations. These markers fail to distinguish between the human infective (T. b. rhodesiense) and nonhuman infective (T. b. brucei) "subspecies" so that parasites derived from hosts other than humans potentially contain both subspecies. To overcome some of the inherent problems with the use of such markers and diverse populations, we have analyzed a well-defined population from a discrete geographical location (Busoga, Uganda) using three recently described minisatellite markers. The parasites were primarily isolated from humans and cattle with the latter isolates further characterized by their ability to resist lysis by human serum (equivalent to human infectivity). The minisatellite markers show high levels of polymorphism, and from the data obtained we conclude that T. b. rhodesiense is genetically isolated from T. b. brucei and can be unambiguously identified by its multilocus genotype. Analysis of the genotype frequencies in the separated T. b. brucei and T. b. rhodesiense populations shows the former has an epidemic population structure whereas the latter is clonal. This finding suggests that the strong linkage disequilibrium observed in previous analyses, where human and nonhuman infective trypanosomes were not distinguished, results from the treatment of two genetically isolated populations as a single population.
Renal-cell carcinoma (RCC) is one of the human cancers which respond best to immunotherapy. To better characterize the mechanism of the immune response in RCC, we analyzed the T-cell receptor (TCR) beta-chain repertoire in primary RCC, metastases and paired peripheral blood lymphocytes (PBL) from 9 patients. For 3 of these, we analyzed T cells recovered from normal kidney, or from tumor-involved lymph nodes as well as tumor-infiltrating lymphocytes (TIL) expanded in vitro for adoptive immunotherapy. The initial semi-quantitative RT-PCR method for definition of the Vbeta gene usage was not informative enough to distinguish intratumoral clonal T-cell expansions. In contrast, the length pattern analysis of the complementary determining regions 3 (CDR3) allowed oligoclonal T-cell populations to be detected in fresh TIL form the 9 patients with RCC. Furthermore, these oligoclonal TIL populations were not present in normal renal tissue, autologous PBL or tumor-involved lymph nodes. Different clonal T-cell expansions were identified in the primary tumor and in a pulmonary metastasis from the same patient. The detection of clonal T-cell populations observed in RCC suggests an in situ expansion in response to potential tumor antigens. This report provides an overall and accurate description of the T-cell repertoire in a significant number of samples from patients with RCC.
Recent polymerase chain reaction (PCR)-based studies focused on the detection of immunoglobulin heavy chain gene (IgH) rearrangements have suggested that clonal populations may be amplified more easily from certain categories of B-cell neoplasia than others and that primer makeup can be a critical factor in successful amplification. However, these particular reports contained relatively few low grade B-cell lymphoproliferative disorders of nonfollicular center cell type (LG-BLPD) and used only a limited panel of available primer sets for PCR amplification of monoclonal B-cell populations. To address this issue more extensively we evaluated 156 samples of LG-BLPD by the PCR to determine optimal primer selection in this setting. All cases were classified according to standard morphological and immunophenotypic criteria, with monoclonality documented by Ig light chain restriction analysis. The LG-BLPD included 33 cases of chronic lymphocytic leukemia (CLL), 57 cases of small lymphocytic lymphoma (SLL), 10 cases of atypical CLL, 32 cases of mantle cell lymphoma (MCL), 17 plasma cell neoplasms (PCNs), and seven cases of hairy cell leukemia (HCL). All primer sets included a 3' IgH joining region consensus primer, whereas the 5' IgH variable region (VH) primer was different in each set. The first-line panel included the following: Set 1, VH-framework III consensus primer, and Set 2, seven separate VH-framework I family-specific primers. A reserve panel of alternate VH consensus primers directed at framework II or III regions was used only when Set 1 showed no evidence of B-cell monoclonality.(ABSTRACT TRUNCATED AT 250 WORDS)
Of the approximately 1,000 odorant receptor (OR) genes in the mouse genome, an olfactory sensory neuron (OSN) is thought to express one gene, from one allele. This is reminiscent of immunoglobulin and T-cell receptor genes, which undergo DNA rearrangements in lymphocytes. Here, we test the hypothesis that OR gene choice is controlled by DNA rearrangements in OSNs. Using permanent genetic marking, we show that the choice by an OSN to express an allele of the OR gene M71 is irreversible. Using M71-expressing OSNs as donors for nuclear transfer, we generate blastocysts, embryonic stem (ntES) cell lines and clonal mice. DNA analysis of these cell lines, whose genome is clonally derived from an M71-expressing OSN, does not reveal DNA rearrangements or sequence alterations at the M71 locus. OSNs that differentiate from ntES cells after injection into blastocysts are not restricted to expression of M71 but can express other OR genes. Thus, M71 gene choice is irreversible but is reset upon nuclear transfer, and is not accompanied by genomic alterations.
The clonality pattern of immunoglobulins (Igs) produced after allogeneic bone marrow transplantation (BMT) was studied by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of serum samples and purified Igs. With this technique, the light (L) chain of a monoclonal Ig usually appears as a single spot. Thus, the degree of clonal diversity of the functional B cells can be appreciated by the electrophoretic pattern of the serum L chains. Furthermore, 2D-PAGE allows a semi-quantitative determination of prominent Ig clones according to the size of L chain spots. We found that serum electrophoretograms of 8/19 patients after BMT (5-9 months) revealed L chain patterns which were similar to those of normal polyclonal Igs, that is, less than five distinguishable small spots among a cloud-like indiscrete L chain spots region ('polyclonal' pattern). A spectrum of clonal abnormalities was observed on the electrophoretograms of 11/19 patients: in five patients, multiple small L chain spots (corresponding to Ig concentrations between 0.2 and 2 g/l) were detected ('oligoclonal' pattern), whereas in six others, 'typical' monoclonal Igs (Ig concentrations > 2 g/l) were observed with (3/19 patients) or without (3/19 patients) multiple small clonal components. Sequential analysis of serum obtained from patients at different times after BMT revealed that imbalanced clonal reconstitution was transient and evolved towards apparently normal polyclonal Ig production. Our observations show that the development of clonal 'gammopathies' after BMT is a frequent, but not obligatory phenomenon. It may reflect a transient restriction of the B cell repertoire either through a limited outgrowth of precursor cells or through selective antigenic pressures.
BACKGROUND: It has been suggested that multiple sites of epithelial ovarian carcinoma on the peritoneal surface reflect polyclonal disease arising from multiple primary tumors in the peritoneal mesothelium, rather than monoclonal disease spread by metastases from one primary ovarian cancer. PURPOSE: The purpose of this study was to investigate whether ovarian cancer has a monoclonal or polyclonal origin. METHODS: DNA specimens were obtained from peripheral blood lymphocytes (normal DNA) and from multiple tumor deposits of 17 women with epithelial ovarian carcinoma: primary tumors, metastatic deposits, and ascites. The clonal origin of each tumor was determined by performing (a) analysis to detect loss of heterozygosity at five loci on chromosomes 5, 11, 13, and 17; (b) sequencing of exons 5-8 of the p53 gene; and (c) X-chromosome inactivation analysis of the phosphoglycerate kinase (PGK) gene. RESULTS: In 15 of the 17 cases analyzed, there was clear evidence of monoclonal origin. The probability that the genetic events documented in these 15 cases occurred as independent events in each tumor deposit ranged from 2.5 x 10(-1) to 3.7 x 10(-16). In two cases, the pattern of allelic deletion and p53 gene mutation was compatible with either a monoclonal origin or origin from two primary ovarian tumors. CONCLUSIONS: The results did not support the hypothesis that ovarian cancer is a multifocal, polyclonal disease. Instead, the data suggest that sporadic epithelial ovarian carcinoma has either a monoclonal or a dual primary origin. IMPLICATIONS: These findings have important implications for understanding of the natural history of ovarian cancer and for clinical strategies aimed at prevention and early detection. Further studies will be required to determine the clonal origin of familial hereditary ovarian cancer.
The NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor cDNA was stably transfected into Chinese hamster ovary (CHO) cells. Northern analysis revealed 3 clonal cell lines expressing high levels of NMDAR1 mRNA (BA1, BA2 and BA3). NMDAR1 protein was readily detected by Western analysis in only one of the clonal populations, BA1. Whole-cell patch clamp analysis revealed a lack of functional NMDA receptors in all of the cell lines, including BA1. NMDA-mediated responses were obtained only after a second subunit, NMDAR2A, was transiently expressed in BA1 cells. Therefore, the NR1 subunit alone may not be sufficient to form functional NMDA receptors in CHO cells.
Inflammatory myofibroblastic tumours (IMTs) were initially considered to be benign reactive processes, but cases with an unfavourable outcome have been reported. Moreover, clonal genetic alterations have recently been published in some cases, suggesting that IMT may represent a malignant neoplastic entity. This paper reports a case of IMT that developed in the mammary gland, an unusual site. The histological picture was characterized by a proliferation of spindle cells with little cellular atypia and rare mitoses, associated with a polymorphous inflammatory infiltrate. Their immunophenotype, characterized by the expression of vimentin, smooth muscle actin, and cytokeratins, corresponded to that of myofibroblasts. Cytogenetic analysis revealed the clonal nature of the lesion. The modal karyotype was 48, X, ins(2;X)(q34;p21.2p22.2), +7, del(9)(p23), +19. Including the present observation, a 9p deletion has now been found in three cases of IMT. These observations show that IMT may be a clonal neoplasm, even in sites different from deep soft tissues.
The use of numerical analysis of macrorestriction profiles resolved by pulsed-field gel electrophoresis (PFGE) for identifying epidemiological and clonal relationships between strains of Salmonella typhimurium was investigated. A cluster analysis based on Xba1 macrorestriction profiles of 40 strains was compared to biotype, phage-type and ribotype data on each isolate to validate the integrity and significance of the results. A significant correlation between these data was noted, suggesting that evolutionary divergence, of both recent and a more distant nature, may be detected. Moreover, two principal clonal lines of Salm. typhimurium appear to be prevalent in Denmark, of which one is comparatively stable with respect to phenotype and genotype, while the other is relatively diverse in these respects. We conclude that numerical analysis of macrorestriction profiles is a useful means of evaluating interstrain relationships between Salm. typhimurium strains and has practical applications for both short- and long-term epidermiological surveillance purposes.
Multiple brain tumors of diffuse large B cell lymphoma (DLBL) were observed in a 75-year-old man with Waldenström's macroglobulinemia (WM). Paravertebral and multiple subcutaneous nodules occurred in succession and he died 4 months after onset. We investigated B cell monoclonality by means of polymerase chain reaction (PCR) analysis and sequencing of the immunoglobulin heavy chain of paraffin-embedded sections. The PCR product of the brain tumors showed a different rearrangement pattern from those of the other sections. The co-occurrence of DLBL with WM is rare, and some investigators have examined the clonality of the two malignancies. This case is important because DLBL brain tumors co-occurred with WM, enabling us to prove that DLBL and WM have different clonality.
SUMMARY: Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety in methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. Using Mycobacterium tuberculosis, a clonally evolving bacterium with a small accessory genome, as a model system, we systematically evaluated sources of variability in pan-genome estimates. Our analysis revealed that differences in assembly type (short-read versus hybrid), annotation pipeline, and pan-genome software, significantly impact predictions of core and accessory genome size. Extending our analysis to two additional bacterial species, Escherichia coli and Staphylococcus aureus, we observed consistent tool-dependent biases but species-specific patterns in pan-genome variability. Our findings highlight the importance of integrating nucleotide- and protein-level analyses to improve the reliability and reproducibility of pan-genome studies across diverse bacterial populations. AVAILABILITY AND IMPLEMENTATION: Panqc is freely available under an MIT license at https://github.com/maxgmarin/panqc.
In order to develop an ultimate therapy to overcome autoimmune diseases, it is very important to identify whether or not autoantigen-reactive T cells accumulate in such diseases. A novel method was established to analyze T cell clonality using a combination of reverse transcriptase-polymerase chain reaction of T cell receptor beta chain transcripts and single-strand conformation polymorphism (RT-PCR/SSCP). Using RT-PCR/SSCP, clonal expansion of T cells were identified in NOD mouse, collagen induced arthritis model, rheumatoid arthritis, Crohn's disease, Sjögren's syndrome, bone marrow transplantation, pregnancy, malignant tumors, HTLV-I associated myopathy, and interstitial pneumonia. By using a tandem mass spectrometry or a lymphocytic proliferation assay, T cell epitopes were mapped in NOD mice, EAE, MCTD, and melanoma.