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Non-random chromosome gains in human lymphoblastoid cell lines
Abstract
HUMAN lymphoblastoid cell lines derived from the peripheral blood
lymphocytes of healthy donors are commonly diploid when examined in the
early months after establishment but acquire chromosome abnormalities on
prolonged culture. Other lines, notably those derived from Burkitt's
lymphoma tissue, may display chromosome aberrations from the
outset1-5. A partial translocation 8q-14q+ has been
demonstrated in the majority of Burkitt lymphoma-derived
lines5,6 and data on the karyotypes of some human tumours
suggest that non-random gains and/or losses of chromosomes may be a
feature, in particular, of certain leukaemias and
lymphomas7-13. As the emergence of an aneuploid clone from a
previously diploid lymphoblastoid line may be associated with other
changes suggesting the development of a more `malignant'
phenotype14, it is relevant to compare the chromosome
aberrations detected in such lines with the human tumour data. We have
therefore undertaken a study of banded karyotypes of eighty EB
virus-carrying human lymphoblastoid lines. The first stage of this
analysis is concerned only with gains and losses of whole chromosomes or
chromosome arms and the data presented here establish that, considering
all the lines together, there have been nonrandom gains of five
autosomes (numbers 3, 7, 8, 9 and 12) and of the sex chromosomes.
... Lymphoblastoid cell lines (LCLs), conducted through in vitro infection of human B-lymphocytic cells with Epstein-Barr virus (EBV), are a well-established tool for preserving patient material with specific genetic aberration(s) for future studies. Although reports as early as 1977 1 indicated that prolonged culture of LCLs resulted in chromosomal gains (specifically for #3, #7, #8, #9, and #12 and for the gonosomes), it continued to be widely reported that LCLs remain karyotypically stable during culture. 2 Furthermore, a 1994 publication 3 demonstrated that EBV transformation results in immortalization in only a small percentage of LCLs, that is, in those which successfully activate telomerase. 4 The majority of LCLs should therefore be considered as "EBV transformed cell lines" rather than immortalized. ...
... In other investigations of EBV-induced LCLs, trisomy 12 was among the numerous rearrangements observed, but not as frequent as in our experiments. 1,7,24 It is very likely that at least under in vitro conditions, clones with trisomy 12 have a higher growth potential compared with those with normal karyotypes. ...
To preserve material for future genetic studies, human B-lymphocytes from whole blood samples are routinely transformed into lymphoblastoid cell lines (LCLs) by in vitro infection with Epstein–Barr virus. To determine the rate and frequency of chromosomal changes during long-term culture, we established 10 LCLs (from eight individuals). Before transformation, these cases showed a normal karyotype (three cases), a small supernumerary marker chromosome (three cases), or an aberrant karyotype (four cases). Chromosome analyses were performed at 8-week intervals over a period of at least 1 year, up to 3 years. Surprisingly, we demonstrate that chromosomal instability is the rule, rather than the exception, during long-term culture of LCLs. The most commonly observed acquired clonal aberration was trisomy 12, which emerged in all cell lines within 21 to 49 weeks after infection. Telomeric fusions indicating telomere shortening were found after ~21 weeks. After 1 year of cultivation, the proportion of cells with the original karyotype decreased to ≤10% in 7 of the 10 cell lines. To preserve cells with aberrant genomes, we conclude the cultivation time of LCLs must be restricted to the absolute minimum time required:
... A pseudotetraploidy of the human genome was found, with a number of chromosomes ranging between 91 and 93. The parental RJ 2.2.5 human Ia-cells presented a chromosome number of 46, with various chromosome abnormalities including t(8; 14) and trisomy 7 (data not shown), frequently found in human Burkitt lymphoma lines (10). The human pseudotetraploidy of A2.2-4 c.6 probably arose by an accidental tri-parental mating, as suggested by the fact that all clones derived from the hybrid A2 exhibited similar human chromosome complement. ...
RJ 2.2.5 is a human B cell line that has lost the capacity to express MHC class II genes. The human class II-positive phenotype is restored in somatic cell hybrids between RJ 2.2.5 and mouse spleen cells. By karyotype and molecular studies of an informative family of hybrids we have now shown that the reexpression of human class II gene products, as well as the maintenance of the mouse class II-positive phenotype, correlates with the presence of mouse chromosome 16. Thus, the existence on this mouse chromosome of a newly found locus, designated by us aIr-1, that determines a trans-acting activator function for class II gene expression, is established. Possible implications of this finding are discussed.
... Hyperdiploidy. on the other hand, was concluded to be a nonrandom event and probably a consequence of non-disjunction. However, in the case of MCL-5 cells we believe that hyperdiploidy is accounted for by duplication and not non-disjunction, as indicated by the presence of two isochromosomes [ Steel etal., 1977 Shade et al.. 1980; Nilsson, 1992). The work of Steel et al. (1977 Steel et al. ( , 1980) and Shade etal. ...
MCL-5 cells are Epstein Barr virus-transformed human lymphoblasts which have been genetically engineered for use in mutagenicity testing. We have examined the modal chromosome number, karyotype and spontaneous micronucleus (MN) and sister chromatid exchange (SCE) frequencies of the cell line. Replicate experiments were conducted on two different shipments purchased from Gentest Corp.Although the modal chromosome number was 48 (range40-54, n = 400 metaphases) for both cell shipments, the second stock showed greater variation in chromosome number than the first.A total of 60 G-banded metaphase cells was analyzed and seven karyotypes were prepared.Consistent structural abnormalities (translocations, deletions and isochromosomes) were found involving the X chromosome and seven autosomes (1-3, 5, 6, 9 and 11).The karyotype typical of this cell line was: 48,der(X)t-(X;?)(P223;?)Y,t(l;2)(q23;p23),del(3)(ql2q21), + i(3q),t(5;6)(q31;p23), + i(9p),der(ll)t(ll;13)(q23,ql2).The mean MN frequency was 41.8 MN/1000 binucleate cells (n = 5000).When compared with our historical controls for primary lymphocyte cultures this number (41.8) is significantly(8.4-fold) higher. The mean SCE frequency was 13 per metaphase (n = 100).We observed a hyperdiploid chromosome number of 48 in the majority of metaphase spreads, indicating a significant deviation from the normal diploid number characteristic of the parent cells (RPMI 1788)established in 1969.The variation in chromosome number distribution observed between shipments suggests the potential for further changes. The elevated MN frequency suggests that evaluating mutagenicity using this cytogenetic end-point may require excessive dosing to produce a significant response over background.We conclude that careful interpretation of cytogenetic end-points is necessary when using MCL-5 cells in the light of the possibility of clonal evolution presented here.
... A pseudotetraploidy of the human genome was found, with a number of chromosomes ranging between 91 and 93. The parental RJ 2.2.5 human Ia-cells presented a chromosome number of 46, with various chromosome abnormalities including t(8; 14) and trisomy 7 (data not shown), frequently found in human Burkitt lymphoma lines (10). The human pseudotetraploidy of A2.2-4 c.6 probably arose by an accidental tri-parental mating, as suggested by the fact that all clones derived from the hybrid A2 exhibited similar human chromosome complement. ...
RJ 2.2.5 is a human B cell line that has lost the capacity to express MHC class II genes. The human class II-positive phenotype is restored in somatic cell hybrids between RJ 2.2.5 and mouse spleen cells. By karyotype and molecular studies of an informative family of hybrids we have now shown that the reexpression of human class II gene products, as well as the maintenance of the mouse class II-positive phenotype, correlates with the presence of mouse chromosome 16. Thus, the existence on this mouse chromosome of a newly found locus, designated by us aIr-1, that determines a trans-acting activator function for class II gene expression, is established. Possible implications of this finding are discussed.
... There may be advantages in employing more stable clones such as C14 and C14/7 that are tetraploid, yet sensitive to glucocorticoids, although there appears to be differences in the kinetics of their responses compared with diploid clones (compare lag periods C14 and C14/7 with C7A, B, C (Figure 3). Other workers have noted chromosomal gains in HLCL in continuous culture (Steel et al., 1977), and although we measure DNA content profiles rather than karyotypes, it may be that what we see as a tendency to aneuploidy, followed by doubling in DNA content, is equivalent to non-random gains in chromosomal numbers. Such gains may be associated with reversion to the resistant state as in the parent CCRF/CEM line. ...
We have found a relationship between sensitivity to glucocorticoid induced cell death (at 10 microM glucocorticoid) and ploidy in the human lymphoid cell line CCRF/CEM-C7. Most sensitive clones are diploid, whilst resistant clones and the resistant parent line CCRF/CEM are tetraploid. Diploid sensitive clones have a tendency to become aneuploid within a few months of isolation, with alterations in their kinetic responses to glucocorticoids. This is followed by a doubling in DNA content which results in reversion to the tetraploid glucocorticoid resistant state of the parent line CCRF/CEM. A few sensitive clones have been found to be tetraploid but with different kinetic responses to glucocorticoids as compared to diploid clones. The principal difference being an extended lag period (48-72 h) prior to lethal response. The relationship between ploidy and glucocorticoid sensitivity does not appear to extend to other human lymphoid cell lines.
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... The most reasonable interpretation is that a B-lymphocyte with what appears to be a normal karyotype was transformed by the virus and became the progenitor for each of the two lines, and that chromosome rearrangement occurred subsequently in one of that progenitor-cell's progeny. Ordinarily, the emergence in LCLs derived from the blood of normal persons of clones with abnormal chromosome complements occurs only after a much longer period of proliferation ([15] and our own unpublished observations). The question arises, then, as to whether the chromosome complements of WS cells are hypermutable. ...
Each of several cultures of Werner's syndrome (WS) fibroblasts and lymphoblasts examined was found to be composed of one or several clones of cells with mutated chromosome complements. Two "sister" fibroblasts cell lines (FCLs) that were derived from a mixture of explants cut from the same WS skin biopsy were found to have completely different rearranged chromosome complements. Daily observation of the skin explants from which these two sister FCLs were derived revealed not only that no more than a few fibroblasts ever migrated from a given explant but also that fibroblasts migrated from only a few of the explants. Two of three lymphoblastoid cell lines (LCLs), each probably developed as an independent clone from a different cell from the same WS blood sample, were mosaic, comprised of cells having both normal and rearranged chromosome complements. The third LCL studied, although nonmosaic, had a rearranged chromosome complement, but one that was completely different from those in the other two lines. Based on the observations described, hypotheses have been formulated to explain both the preponderance in long-term WS cultures of clones with mutated chromosome complements and the abbreviated lifespan characteristic of WS fibroblast cultures.
... It is not yet clear whether the heterogenicity in repair capacity existing among different cell lines points to a true genetic polymorphism present in human populations. It may reflect a change in a certain regulatory mechanism which occurs follow ing transformation of lymphocytes by Epstein-Barr virus, or it may be the result of a selection process taking place at the stage of cell transformation (39) or during prolonged mainte nance in culture (24,40). It may be analogous to the reduction in cellular capacity to repair O6-MeG, which was found in certain tumor strains (5) and resulted in hypersensitivity to MNNG. ...
Human lymphoblastoid cell lines from normal individuals and from patients with ataxia telangiectasia were either proficient or deficient in their ability to repair the mutagenic DNA adduct O6-methylguanine that is induced by methylating carcinogens. There was no relationship between the capacity to repair O6-methylguanine and the ataxia telangiectasia phenotype. Time-course studies done following a short pulse (2 min) of alkylation with 0.5 microgram of N-[3H]methyl-N'-nitro-N-nitrosguanidine per ml revealed that the repair of O6-methylguanine in human lymphoblastoid lines proficient in this ability is a rapid process, which proceeds with a half-life of 10 to 15 min. Lymphoblastoid lines with deficient capacity to repair this DNA adduct were hypersensitive to the cytotoxic effect of the methylating carcinogens N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, and methyl methanesulfonate, and this hypersensitivity was correlated with the relative amount of O6-methylguanine induced by each of the three chemicals. This was taken as an indication of the lethality of unrepaired O6-methylgluanine. The extent of DNA repair synthesis induced by the three carcinogens was the same in cell lines proficient and deficient in O6-methylguanine repair, indicating no major deficiency in an excision repair pathway in the hypersensitive cell lines.
Lymphoid cell lines have been invaluable tools not only in studies of the biologic properties of Epstein-Barr virus (EBV), but also in the fields of oncology, hematology, immunology, genetics, and cell biology. In 1964, Pulvertaft (1964 a) and Epstein and Barr (1964) independently described the first cell lines from tumor biopsies of patients with Burkitt’s lymphoma (BL). Before this, attempts to cultivate normal and malignant hematopoietic tissue in vitro had usually led to a gradual deterioration and subsequent death of the various cell types within weeks or months (Osgood, 1958; Reisner, 1959). Only rarely had permanent cell lines been established from leukemic blood (Osgood and Broke, 1955) and bone marrow (Benyesh-Melnick et al., 1963). In the latter report, the sudden outgrowth of permanent cell lines with a lymphoblastoid morphology was described at a low frequency in fibroblastoid monolayers several weeks after initiation of bone-marrow cultures derived from children with leukemia, infectious mononucleosis (IM) and hemolytic anemia. This remarkable event was termed “lymphoblastoid transformation of fibroblastic bone-marrow cultures.” The meaning of the term “lymphoblastoid transformation” here was thus different from the “lymphoblastoid transformation” used to describe the morphologic changes of normal lymphocytes after exposure to phytohemagglutinin (PHA) (Nowell, 1960).
Six acetylenic compounds were isolated from the leaves of Artemisia lactiflora (Compositae), an edible plant of Thailand. Four of them were identified as the stereoisomers of 3,4-epoxy-2-(2,4-hexadiynylidene)-1,6-dioxaspiro[4.5]decane and its derivatives, whose planar structures have already been reported. The other two were identified as novel chlorohydrin derivatives of the corresponding diacetylene spiroketal enol ethers. The inhibitory effects of the polyacetylenes isolated in the present study together with genistein on TPA-induced O2- generation were examined. It was revealed that an acyloxyl group at the C-2 position enhanced the inhibitory effect, while the absolute configurations at C-5, -6, and -7 were not important. While polyacetylenes are known to possess several biological roles in various ecosystems, we first found inhibitory effects of the diacetylenes on TPA-induced O2- generation in differentiated HL-60 cells. Keywords: Artemisia lactiflora; superoxide; HL-60; diacetylene spiroketal enol ether; tumor promoter; cancer chemoprevention
Rosmarinic acid (RoA) has been isolated in high yield (0.1%) from the leaves of Perilla frutescens Britton var. acuta f. viridis, one of the most common garnishes in Japan, as a superoxide scavenger in a xanthine/xanthine oxidase system. The scavenging activity of RoA was significantly higher than that of ascorbic acid (AA). The structure−activity relationships using RoA, caffeic acid (CaA), and its related compounds indicated that the presence of an ortho dihydroxyphenyl group is essential for the scavenging effect. In addition, the conjugated double bond in the C3 carbon chain is important for enhancing the effect. The activity of RoA was then shown to be due to the additive effect of both CaA and (3,4-dihydroxyphenyl)lactic acid units, both of which are hydrolyzed products of RoA. RoA significantly inhibited both intracellular superoxide and peroxide formation in differentiated HL-60 cells, suggesting that RoA effectively exhibited antioxidative activity in the biological systems through the scavenging of superoxide. Keywords: Perilla frutescens; rosmarinic acid; superoxide; xanthine oxidase; HL-60
Summary We describe a novel continuous B-cell line (PV-90) derived from a patient with myelodysplastic syndrome (MDS) and originating from spontaneous infection with the Epstein-Barr virus (EBV). The patient progressed to acute myeloblastic leukaemia (AML) 5 months after clinical onset of MDS. PV-90 is of clonal origin as indicated by the presence of immunoglobulin (Ig) gene rearrangements, monoclonal surface immunoglobulins, and a single DNA restriction fragment corresponding to the EBV genomic termini. PV-90 cells also express a number of myelomonocytic markers, including α-naphthyl acetate esterase (ANAE), coagulation factor XIII, and CD68 antigen. Moreover, PV-90 cells constitutively express the c-fms proto-oncogene mRNA as the patient's blast cells did. Whereas a trisomy 11 (+11) was found in the patient's bone marrow cells, PV-90 cells had a normal karyotype initially, but at 4 months showed two different and independent chromosomal abnormalities: 90, XX, -Y, -Y, t(9;16) (q11;p13), and 90, XX, -Y, -Y, t(17;18) (p13;q21), the latter possibly involving the p53 (17, p13) and bcl-2 (18, q21) proto-oncogenes. The early development of these chromosomal aberrations is consistent with a genetic instability of PV-90 cells. Expression of bi-lineage markers and genetic instability may suggest that PV-90 cells originated from transformation of a myelodysplastic progenitor cell capable of both myeloid and B-cell differentiation. The PV-90 cell line might be useful in a number of studies, including the possible role of c-fms in cell differentiation, pathogenetic mechanisms of human preleukaemia and lineage promiscuity in acute leukaemia.
The observation that decreased thymidylate supply in vitro induces the expression of the Xq27 chromosome fragile site prompted us to examine cellular thymidylate metabolism. Using a sensitive enzyme assay for deoxyribonucleotide triphosphates, we found that the total cellular thymidine triphosphate pools in cell lines from fragile X patients and carriers do not differ from normal controls under either basal or folate-deficient conditions. This agrees with our earlier observation that the thymidylate synthase enzyme activities in crude cell extracts of five fragile X syndrome lymphoblast lines do not differ from those in normal controls under standard assay conditions (1). Although a difference in the amount of thymidine triphosphate available at the replication fork for DNA synthesis remains a possibility, our results indicate that a readily demonstrable defect in thymidylate metabolism is not present in fragile X syndrome cells.
Epstein-Barr-virus-associated posttransplant lymphoproliferative disease ranges from transient lymphadenitis to aggressive
lymphoma. This study characterizes an in vitro model to study the pathogenesis of this disease with a cell culture system. Five B-cell lines derived from posttransplant
lymphoproliferative disease tissue were characterized with regard to immunophenotype, karyotype, molecular genetics, cytokine
production, and growth regulation. All cell lines expressed CD19, CD21, CD22, CD43, and CD77, but not CD10 antigens. Immunoglobulin
light chain restriction was seen in four of five cell lines, and cytogenetic abnormalities were demonstrable in three of the
five. Cells proliferating in culture contained multiple Epstein-Barr virus episomes and showed lytic viral replication. All
cell lines produced tumor necrosis factor-β and interleukin-10 without evidence of autocrine growth regulatory loops involving
these cytokines. No evidence of IL-1 alpha, IL-2, IL-4, IL-5 or IL-6 production was found by reverse transcriptase polymerase
chain reaction. Adding 500 U IFN-α/ml to the culture medium resulted in 30% inhibition of [3H]thymidine incorporation.
The E2F transcription factor plays an important regulatory role in cell proliferation, mediating the expression of genes whose products are essential for inducing resting cells to enter the cell cycle and synthesize DNA. To investigate the possible involvement of E2F in hematopoietic malignancies, we isolated genomic clones encompassing the human E2F1 gene. We then used fluorescence in situ hybridization to localize E2F1 to human chromosome 2011, telomeric to the p107 locus, a gene whose product is related to the retinoblastoma gene product (pRb). This finding contrasts with the 1p36 and 6q22 chromosomal locations previously assigned E2F2 and E2F3, two additional members of the E2F family. Although deletions or structural rearrangements of E2F1 were not detected in 14 primary acute leukemia or myelodysplasia samples with structural abnormalities of chromosome 20q11, the gene was amplified and overexpressed in HEL erythroleukemia cells and translocated to other chromosomes in several established human leukemia cell lines. This study provides the first evidence of gene amplification involving a member of the E2F family of transcription factors. We propose that E2F1 overexpression in erythroid progenitors may stimulate abnormal cell proliferation by overriding negative regulatory signals mediated by tumor suppressor proteins such as pRb.
We present high-resolution 8–14 μm observations of Shoemaker–Levy 9 sites conducted on July 20, 30, and 31 1994 UT at the NASA Infrared Telescope Facility. Stratospheric heating was detected from strong enhancements of methane emission near 8.1 μm over areas at least 15,000 km wide around the K site observed 23 hr after impact and around the L site 11 hr after impact. The intensity distribution between strong and weaker CH4lines implies that the stratospheric heating was primarily confined to pressures less than 500 μbar. The L site temperature increased by 80 ± 10 K at 5 μbar, but did not exceed 20 K around 1 mbar or 10 K around 10 mbar. The older K site was still 30 ± 5 K warmer than the surroundings at the 10-μbar level. The excess thermal energy stored in the upper jovian stratosphere was 3+3−1.5× 1026erg over the L site, and 2+2−1× 1026erg over the K site at the time of the observations. Comparison with numerical simulations indicates that a large fraction (>20%) of the kinetic energy of the L plume was transferred to the jovian atmosphere and not immediately radiated away. Acetylene line emission near 13.4 μm was enhanced over an area ∼18,000 km wide centered on the E site 2.6 days after impact. Radiative transfer models of this emission indicate temperatures 37 ± 7 K higher than nominal around 3 μbar. No such enhancement was seen in CH4spectral images, implying that the temperature perturbation did not significantly extend below the ∼20-μbar level. The H site observed simultaneously was 12 ± 5 K warmer than the surroundings 1.4 day after impact. C2H2lines were still slightly more intense over the K + W and Q1 sites on July 30 and 31, 8 to 10 days after impact. These observations can be interpreted either by temperature differences of about 13 and 10 K respectively in the 3-μbar region, or by an increase in the C2H2column density of 2.5–5 × 1017molecule cm−2. Emission from hydrogen cyanide lines around 13.4 μm was detected over all sites observed. The mass of HCN is about 2 × 1012g for the biggest plumes (K, L, G), 0.95 ± 0.5 × 1012g over the E site, and 0.45 ± 0.2 × 1012g over the H site. The total mass of HCN produced by all fragments is estimated to be 1.1 ± 0.4 × 1013g. A consistent interpretation of the different pieces of information available suggests that the H $cl10$plume was richer in dust than the E or A plume.
Cell lines were established from the fresh peripheral blood of over 50% (11 of 21) of Epstein-Barr virus (EBV) seropositive human donors after exposure to viruses of the gibbon ape leukemia virus (GALV)—simian sarcoma virus/simian sarcoma-associated virus (SiSV/SiSAV) group. The incidence of growth induction upon exposure to baboon endogenous virus (BaEV), feline leukemia virus (FeLV) or to uninfected monolayer cultures was lower than 10%. Leukocytes from the peripheral blood of leukemic patients gave similar results, but exposure of fresh peripheral blood from eight normal EBV seronegative donors to the same viruses did not result in induction of cell growth. The established cell lines were polyclonal B-lympho-blasts as determined by morphological cytochemlcal, immunological and functional properties. The cells expressed EBV nuclear antigen (EBNA test), formed rosettes with complement-activated sheep or bovine erythrocytes (EAC-rosette test), but not with untreated erythrocytes (E-rosette test), and were positive for one or more surface-bound immunoglobullns. They were also karyotypically normal as determined by standard chromosome analysis and by trypsin-Giemsa banding techniques. However, unlike other reported newly-established diploid B-lymphoblast cell lines, cells from approximately 80% of the cultures tested grew as locally invasive tumors when inoculated into 1-to 3-week-old athymic nude mice and formed colonies in semi-solid medium. Lymphoblasts from nude mouse tumors were readily re-established as suspension cultures which retained their lymphoblastic properties and remained karyotypically normal. These studies indicate that under certain conditions, type-C retroviruses of the GALV-SISV/ SISAV group directly or indirectly increase the incidence of transformation of human B lymphocytes.
THE incidence of lymphomas is 10,000 times greater than normal in patients with immunodeficiency syndromes1 and also in ataxia telangiectasia (AT), which very often involves IgA deficiency2. In addition to its other characteristic features, AT is usually associated with a defect in DNA repair3 and a translocation involving chromosome 14 in the peripheral leukocytes4. Another feature is a high prevalence of Epstein-Barr virus early antigen (EBV-EA) antibodies5 in the sera of AT patients. This is significantly higher than in normal human sera (
The in vitro and in vivo growth of lymphoblastoid cell lines (LCLs), Burkitt lymphomas (BLs) and PBL-derived immunoblastomas in SCID mice was studied in parallel. Most, but not all, of the LCLs tested were tumorigenic in SCID mice. Long-term culturing in vitro was not a necessary prerequisite for tumorigenicity. There was a good correlation between in vivo and in vitro growth. Three major classes of cells could be distinguished on the basis of their growth properties.
Mice with severe combined immunodeficiency (SCID) received 6 X 10(7) fresh human peripheral blood mononuclear cells (PBMC) intraperitoneally from Epstein-Barr virus (EBV)-seropositive and -seronegative donors. B95-8 EBV was inoculated intraperitoneally and intravenously to the mice 6 weeks after transfer of seronegative PBMC. Three of four mice transferred with PBMC from two EBV-seropositive donors and two of four mice from two EBV-seronegative donors inoculated with EBV developed fatal EBV-induced lymphoproliferative disease within 6 to 10 weeks. These tumors were oligoclonal or polyclonal by cytoplasmic immunoglobulin expression. Furthermore no consistent clonal chromosomal abnormalities were shown. Cell lines established from these tumors showed low cloning efficiency in soft agarose. In addition, latent membrane protein, B-lymphocyte activation antigen (CD23), and cell-adhesion molecules (ICAM-1, CD18) all were expressed in the EBV-positive infiltrating lymphoproliferative lesions in each mouse. These results suggest that lymphoid tumors are comparable to lymphoblastoid cell lines immortalized by EBV and are not malignant lymphomas such as Burkitt's lymphoma. This model may be useful for investigating mechanisms responsible for the growing numbers of lymphoproliferative diseases that are occurring in patients with inherited or acquired immunodeficiencies.
The non-Hodgkin's lymphomas are a clinically, morphologically, and immunologically heterogeneous group of diseases. Why lymphoma cells are unresponsive to normal regulatory growth controls and how they differ from normal lymphocytes are not well understood. In order to address these questions we have raised monoclonal antibodies to neoplastic B-cells. Two of these, LM-26 and LM-155, show a high degree of specificity for B-cell lymphomas. When tested by fluorescence activated cell sorter analysis, LM-26 reacted with 80% (18 of 23) of B-cell lymphomas freshly explanted from patients and LM-155 reacted with 20% (5 of 23). The antigenic determinant detected by LM-26 was also found to be present on four of seven neoplastic large cell B-lymphoma lines. LM-155 detected a determinant present on all seven of these lines. For neither monoclonal antibody was there any association between antibody reactivity and the morphological subtype of lymphoma examined or the type of cell surface immunoglobulin expressed. LM-155 reacted with one case of B-cell acute lymphoblastic leukemia. Neither antibody reacted with normal B-cell blasts, normal peripheral blood mononuclear or marrow cells, T-cell leukemias or lymphomas, or chronic lymphocytic leukemia cells. Lymphocytes from reactive lymphoid hyperplasias involving lymph nodes, spleen, peripheral blood, and lung were also negative for LM-26 and LM-155 binding or showed only a small percentage of cells positive (4-8%). Both monoclonals were unreactive with non-B-lymphoid neoplastic cell lines, nine of ten Epstein-Barr virus transformed B-cell lines, and cells freshly explanted from patients with cancers of diverse cellular origins. Fluorescence activated cell sorter analysis of the expression of the antigens defined by LM-26 and LM-155 on lymphoma cells and normal B-cell blasts suggests that they are not normal differentiation antigens associated with lymphocyte activation or proliferation. The highly restricted expression of detectable levels of antigens reactive with monoclonal antibodies LM-26 and LM-155 on non-Hodgkin's lymphoma cells suggests a possible relation to their neoplastic properties. From a practical viewpoint these monoclonals may prove useful in the diagnosis, classification, detection of residual disease, and treatment of the non-Hodgkin's lymphomas.
X-linked lymphoproliferative disease (XLP) is triggered by Epstein-Barr virus. This virus is also associated with Burkitt lymphoma, a tumor that carries specific chromosome translocations. No such chromosome translocations have been observed in an analysis of XLP-derived cell lines. One XLP patient was found also to have Klinefelter syndrome, having inherited two copies of his maternal XLP-carrying X chromosome.
Publisher Summary This chapter summarizes the major findings that are significant in understanding the clonal development and progression of human hemopoietic neoplasia. Determination of whether a cellular proliferation is monoclonal or polyclonal has important pathogenetic implications. Polyclonal proliferations are often the result of normal processes in which a tissue responds appropriately to an exogenous stimulus. On the other hand, monoclonal proliferations reflect disordered hemopoiesis in which a clone of cells gains in vivo proliferative advantage. The ability to detect monoclonality in proliferating cells requires a marker system that enables the progeny of different cells to be recognized. Markers that are used effectively to investigate human lymphoid neoplasia include immunoglobulin (Ig) proteins synthesized by relatively mature cells of B-lymphoid origin, DNA sequences coding for these Igs, and T-cell receptor gene rearrangements. Beyond determining whether a disease state is clonal at the time of presentation, studies with cell markers allow characterization of the clonal state throughout subsequent stages of the disease and the differentiative expression of the stem cell involved by the clonal proliferation. Many human hemopoietic neoplasias appear to have a multistep pathogenesis. The diploid cell may represent an early stage in leukemogenesis. Subsequent development of structural chromosomal alterations or aneuploidies may lead to frank leukemia by any of a number of mechanisms, such as activation of an oncogene, amplification of a gene whose product is important for growth, or loss of a gene involved in repressing or regulating growth. The malignant phenotype then progresses by stepwise evolution.
In this brief review we will discuss some recent trends in work on regulatory mechanisms involved in the selective expression of differentiated markers in human B-cell lines as well as the types of controls involved in the expression and function of Epstein-Barr virus (EBV) associated markers in human cells. A problem of special interest in this connection is the relation of the expression of EBV functions to the expression of “transformation”.
This chapter focuses on the recent experimental studies that suggest a different role of Epstein–Barr virus (EBV) in the pathogenesis of Burkitt's lymphoma (BL). Particular emphasis is placed on the distinct phenotypic and cytogenetic differences that have been reported to exist between EBV-transformed normal B lymphocytes and BL cells. They show that the in vitro transformation of B cells cannot be regarded as the full equivalent of the malignant transformation of the BL precursor cell in vivo. The chapter also discusses the secondary abnormal progression of lymphoblastoid cells during continuous growth in vitro, including the acquisition of cytogenetic abnormalities, the potential for tumor formation in nude mice, and high clonability in agarose. Finally, the results are examined in relation to the recent seroepidemiological data concerning the relationship of EBV to the development of BL and the specific chromosomal translocations in BL cells. Furthermore, a BL clone develops from B lymphocytes that have acquired preneoplastic characteristics because of EBV transformation and the fully malignant phenotype develops only when the lymphoblastoid cell acquires a specific chromosomal translocation.
Normal cells become tumorigenic after multiple genetic and epigenetic alterations. This process alters complex signaling networks
within these cells as well as interactions between these cells and the extracellular matrix. Proto-oncogene products tightly
regulate these signaling networks. In this chapter, the signaling pathways through growth factor receptors such as epidermal
growth factor receptor (EGFR) and c-Met are discussed. Expression and activation of these receptors are frequently altered
in human cancers by genetic and epigenetic mechanisms. Activation of these growth factor receptors triggers a cascade of signaling
events that contribute to malignant transformation. Many of the signaling intermediates are themselves proto-oncogenes that
can be activated by growth factor receptor-dependent and-independent mechanisms. Three of these proto-oncogene products, Ras,
Src, and Myc are discussed in relationship to their contributions to malignant transformation.
Two hematologic cases with translocations involving chromosomes #3 and #12 are described. The first case is that of a myeloproliferative disorder (preleukemia?) associated with a (3;12)(q29;q24) translocation in the bone marrow cells. No evidence of leukemic transformation has appeared to date. The second case is that of acute leukemia (AL) (M4 type) in which leukemic cells with t(3;12)(p14;q24) were seen. The roles of chromosomes #3 and #12 in hematopoiesis are considered, and the abnormalities affecting these chromosomes in various hematologic disorders have been tabulated and correlated. Abnormalities in chromosomes #3 and #12 appear to be common and nonrandom in hematologic diseases of a premalignant and a malignant type.
The ability of human fibroblast strains to repair the mutagenic DNA adduct O6-methylguanine (O6-MeG) induced by brief exposure to N-methyl-N'-nitroso-N-nitrosoguanidine (MNNG) was investigated. The repair reaction proceeded rapidly during the first hour after alkylation, followed by a slow, continuous phase of repair, and both processes were saturated by low doses of carcinogen. This was similar to what had previously been found in human lymphoblastoid lines. Three fibroblast strains from healthy donors and six strains from patients with ataxia telangiectasia were all proficient in their capacity to repair O6-MeG and had the same sensitivity to the cytotoxicity of MNNG and methyl methanesulphonate as normal cells. Three of these cell strains were derived from individuals whose lymphoblastoid lines were deficient in their ability to repair O6-MeG. These lymphoblastoid lines were also extremely hypersensitive to killing by methylating carcinogens. Because non-transformed cells from the same donors behaved normally with regard to both parameters, we concluded that the repair deficiency accompanied by carcinogen hypersensitivity of the lymphoblastoid lines does not indicate a genetic deficiency in the donor. These findings imply that lymphoblastoid lines may not always be the appropriate cell type for investigating genetic susceptibility to chemical mutagens.
A 60-year-old man developed pancytopenia and then acute leukaemia. The neoplastic cells in marrow were undifferentiated by electron microscopy and by immunological and cytochemical markers. The only other cells present in marrow were lymphocytes, plasma cells, macrophages and non-haematopoietic elements. Prior to chemotherapy, cytogenetic analysis of marrow cells showed two karyotypically distinct cell populations, one with 45,X,--Y and the other with a 46,X,--Y,+12 karyotype. All marrow cells stimulated by protein-A from staphylococcus aureus were 46,X,--Y,+12. Phytohaemagglutinin-stimulated cells were normal, 46,XY. These findings suggest strongly that most of the undifferentiated leukaemic cells were missing the Y chromosome. A subpopulation of these leukaemic cells also had trisomy 12. These observations and previously published findings suggest that trisomy 12 occurs non-randomly in haematological disorders, and in particular, may be associated with B-lymphoid malignancy.
Human lymphoblastoid cell lines evolve in vitro by the emergence of successive waves of clones which are often chromosomally marked. This offers the opportunity to compare tissue samples of the same genetic origin but differing in certain defined parts of the karyotype.
Using selected sets of lines in which the members of genetically matched pairs differed in the number of copies of 8p or of 12p, levels of GSR and LDH B respectively have been shown to correlate with the specific chromosome aberrations, supporting existing data on the regional assignment of these two structural loci.
This approach represents a useful addition to established methods for human gene mapping.
Isoenzymes determined by 11 loci have been examined in 137 human lymphoblastoid lines of various origins with a view to determining their phenotypic stability in culture. Lines of normal origin are stable and at these loci are phenotypically identical to the individuals from whom they are derived. Lymphomas and some lines from patients with leukaemias show a tendency to increased apparent homozygosity, presumably resulting from loss of expression of one or other allele during culture. Taken together with the cytogenetic evidence this suggests that progressive loss of functional parts of the genome with time in culture is a characteristic of lines derived from malignant lymphoid cells.
S ummary
The occurrence of isozymes of the proteolytic enzyme amino acid naphthylamidase was investigated in a panel of 44 neoplastic and non‐neoplastic haematopoietic cell lines by polyacrylamide gel electrophoresis. Two isozymes (A and C) were common for all lines, whereas different forms of a third isozyme with intermediate electrophoretic mobility (B) appeared in nine lines. Isozyme A seemed to be associated with cell proliferation and was not a marker for malignancy. The appearance of different B isozymes not present in normal peripheral blood lymphocytes, occurred in some Burkitt lymphoma lines and in two non‐malignant lines maintained for a long time in culture. In three lines derived from myeloid leukaemia a variant of isozyme B seemed to be a marker for cell origin.
A B-cell line was established from the liver of an 11-yr-old boy with Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD). The cells were morphologically heterogenous, CD10 (CALLA) negative, and expressed several B-cell antigens, including CD23, in a manner reminiscent of lymphoblastoid cell lines (LCLs) reported in the literature. However, the cells also showed expression of the CD77 antigen, carried a 14q32+ chromosomal anomaly, and showed IgM-kappa immunoglobulin isotype restriction immediately after their outgrowth in culture. These latter properties are typically associated with Burkitt's lymphoma cell lines rather than LCLs. Aberrant expression of the L60 antigen on these B-cells was found as additional evidence of altered growth regulation in these cells. EBV infection was demonstrated by the abundant expression of EBNA-2 and LMP viral antigens in culture. The cell line described should be useful in planning in vitro experiments designed to understand the factors that modulate the growth of PTLD in vivo.
Hodgkin (H) and Reed-Sternberg (RS) cells are considered to be the malignant cell population in Hodgkin's disease (HD). To date, their analysis has been hampered by their scarcity in primary tumors, poor growth in vitro, and lack of an animal model. To establish an in vivo system for the characterization of the malignant cells in HD, tumor biopsy samples from 13 HD patients were transplanted beneath the renal capsule or into the liver of severe combined immunodeficient (SCID) mice. HD-derived tissue from three patients gave rise to human tumors in SCID mice. Three different histologic patterns were observed: (1) lymphoproliferative disease (LPD), (2) anaplastic large cell lymphoma (ALCL), (3) Hodgkin-like lesions (HDLL). Immunohistochemical analysis showed that the tumors consisted of activated B cells (CD30+, CD20+). Epstein-Barr virus (EBV)-encoded transcripts were found in 80% to 100% of the tumor cells, although H and RS cells in the primary tumors of two patients were EBV-. All tumors examined (3 of 3) and the majority (6 of 10) of cell lines recultured in vitro had an abnormal karyotype. Southern blot analysis of the human Ig heavy chain gene showed that monoclonal or oligoclonal tumors of different B-cell origin grew in the SCID mice from the same germ line-configurated primary biopsy specimen. Our data suggest that the human cells in the SCID mice have either been derived from EBV superinfected H and RS cells or from EBV-infected bystander cells. If the latter is true, then these bystander cells must be genetically abnormal. The genetic defect would be either aneuploidy or instable euploidy. In either case, the cells might proliferate into malignant aneuploid HDLL or ALCL under the influence of EBV and the special environment encountered in the SCID mice.
In human HL-60 promyelocytic leukemia cells, diazepinylbenzoic acid derivatives can exhibit either antagonistic or synergistic effects on the differentiation-inducing activities of natural or synthetic retinoids, the activity depending largely on the nature of the substituents on the diazepine ring. Thus, a benzolog of the retinoid antagonist LE135 (6), 4-(13H-10,11,12,13-tetrahydro-10, 10,13,13,15-pentamethyldinaphtho[2,3-b][1,2-e]diazepin-7-yl) benzoic acid (LE540, 17), exhibits a 1 order of magnitude higher antagonistic potential than the parental LE135 (6). In contrast, 4-[5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyldibenzo[b,e] [1,4]diazepin-11-yl]-benzoic acid (HX600, 7), a structural isomer of the antagonistic LE135 (6), enhanced HL-60 cell differentiation induced by RAR agonists, such as Am80 (2). This synergistic effect was further increased for a thiazepine, HX630 (29), and an azepine derivative, HX640 (30); both synergized with Am80 (2) more potently than HX600 (7). Notably, the negative and positive effects of the azepine derivatives on retinoidal actions can be related to their RAR-antagonistic and RXR-agonistic properties, respectively, in the context of the RAR-RXR heterodimer.
The structure-activity relationships of two series of novel retinoids (2-pyrazinylcarboxamidobenzoates and beta-ionylideneacetamidobenzoates) have been investigated by evaluating their ability to induce differentiation in both human promyelocytic leukemia (HL60) cells and mouse embryonal carcinoma (P19) cells. The most active compound (ED50 = 8.3 x 10(-9) M) of the 2-pyrazinylcarboxamidobenzoates is 4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethylquinoxalyl)carboxamido]benzoic acid (9u), while the most active analogue of the beta-ionylideneacetamidobenzoates is 4-[3-methyl-5-(2',6',6'-trimethyl-1'-cyclohexen-1'-yl)-(2E, 4E)-pentadienamido]benzoic acid (10a, ED50 = 3.2 x 10(-8) M). Our studies identify an absolute requirement for the carboxylic acid moiety on the aromatic ring to be para relative to the amide linkage for activity. Benzoate substitutions in the ortho position relative to the terminal carboxylate (9d,k,r) are well-tolerated; however, a methoxy substituent meta relative to the terminal carboxylate gives rise to only weakly active analogues (9x). Conformational studies (NMR, X-ray crystallography) of the 2-pyrazinylcarboxamidobenzoates indicate that the preferred conformation exhibits a trans-amide bond and an internal hydrogen bond between the quinoxaline N1 and HN amide which locks the torsional angle between C2 and CO in the s-trans conformation. N-Methylation (9y) results in loss of activity. Studies indicate that there is now a cis-amide bond present which redirects the carboxylate toward the pharmacophoric gem-dimethyl groups. The distance between the gem-dimethyl group and the terminal carboxylate appears to be too short to activate the retinoid receptor. N-Methylation in the beta-ionylideneacetamidobenzoate series (10c) also results in the formation of a cis-amide bond and loss of activity.
One known, (2R)-(12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dien+ ++-1-yl acetate (1), and two novel compounds, persenone A (2) and B (3), have been isolated from avocado fruit (Persea americana P. Mill), as inhibitors of superoxide (O(2)(-)) and nitric oxide (NO) generation in cell culture systems. They showed marked inhibitory activities toward NO generation induced by lipopolysaccharide in combination with interferon-gamma in mouse macrophage RAW 264.7 cells. Their inhibitory potencies of NO generation (1, IC(50) = 3.6; 2, IC(50) = 1.2; and 3, IC(50) = 3.5 microM) were comparable to or higher than that of a natural NO generation inhibitor, docosahexaenoic acid (DHA; IC(50) = 4.3 microM). Furthermore, compounds 1-3 and DHA markedly suppressed tumor promoter 12-O-tetradecanoylphorbol-13-acetate-induced O(2)(-) generation in differentiated human promyelocytic HL-60 cells (1, IC(50) = 33.7; 2, IC(50) = 1.4; 3, IC(50) = 1.8; and DHA, IC(50) = 10.3 microM). It is notable that they were found to be suppressors of both NO- and O(2)(-)-generating biochemical pathways but not to be radical scavengers. The results indicate that these compounds are unique antioxidants, preferentially suppressing radical generation, and thus may be promising as effective chemopreventive agent candidates in inflammation-associated carcinogenesis.
B cell lymphoma develops in the pleural cavity of patients affected by long-standing pyothorax resulting from lung tuberculosis, thus termed pyothorax-associated lymphoma (PAL). PAL usually shows a diffuse large cell morphology, and constantly contains Epstein-Barr virus (EBV) genome. To investigate whether PAL cells proliferate in response to specific antigenic stimuli and its stage in B cell differentiation, immunoglobulin heavy chain gene in 7 cases and 2 cell lines from PAL, all confirmed by histological studies to be EBV-positive diffuse large B cell lymphoma, were examined by using polymerase chain reaction (PCR) method. Clonal rearrangement of the gene was detected in 4 cases of PAL tissues and one cell line. As for the usage of the V region gene (V(H)), the V(H)3 family gene was used in 3 of these 5 cases with different homologous germlines, suggesting that the origin of PAL cells from a repertoire of B lymphocytes responsive to specific antigenic epitope was unlikely. Compared to the homologous germline, the mutation frequency of PAL was 9% on average. Only one case might have more replacement mutations in the complementarity-determining regions than expected by chance, thus antigen-selected maturation might not take place in PAL. Intraclonal sequence heterogeneity in the V(H) gene was found in another case. From these findings, it is concluded that PAL is composed of B lymphocytes at the differentiation stage of the postgerminal center. Antigen-selected maturation might not take place in PAL, which is distinct from the majority of B cell lymphomas.
The chromosome banding pattern was analyzed in bone-marrow cells and/or spleen cells of 10 patients in the blastic phase of chronic myeloid leukemia (CML). It was obvious from the karyotype analysis that the chromosome aberrations occurring in addition to the Philadelphia chromosome (Ph1) were strictly non-random. An extra Ph1, trisomy 8 and/or trisomy for the long arm of chromosome 17 were observed in all cases. This consistent pattern of chromosome involvement in CML was confirmed in 57 cases from the literature studied with banding techniques. In 88% of the total number of cases with further changes at least one of the three main chromosomal aberrations was found (“major route” of karyotypic evolution).
évolution Caryotypique Non Aléatoire Dans La Leucémie MyéLoïde Chronique
La configuration des bandes chromosomiques a été analysée dans des cellules de moëlle osseuse et/ou des cellules spléniques de 10 malades atteints de leucémie myéloïde chronique (CML) au moment de la transformation blastique. Il ressort de l'analyse caryotypique que les aberrations chromosomiques que l'on a constatées, sans parler de la présence du chromosome de Philadelphie (Ph1), sont strictement non-aléatoires. Dans tous les cas, on a observé un Ph1 surnuméraire, une trisomie 8 et/ou une trisomie du bras long du chromosome 17. Ces altérations chromosomiques que l'on constate régulièrement dans les cas de CML ont été décelées par la technique des bandes dans 57 cas signalés dans la littérature. Dans 88% du nombre total de cas où l'on a observé d'autres altérations, on a constaté au moins une des trois principales aberrations chromosomiques (“voie majeure” de l'evolution caryotypique).
Recent advances in immunology and cell biology have indicated that the diagnosis of a hematopoietic malignancy in many instances should not be based solely on classical morphology. For instance, it has been demonstrated that lymphocyte subpopulations, although morphologically indistinguishable, may have quite different functional properties and obviously represent different stages of lymphoid differentiation. Tumors may evolve from any of these morphologically identical cell types. The position in the differentiation lineage of a malignant cell can be expected to be followed by a unique in vivo behavior, e.g. with respect to growth rate and tendency to metastasize. For therapeutic and prognostic reasons it is therefore essential to delineate the origin of the tumor cell clone by non-morphological methods.
The innovation of chromosome banding methods has affected the entire field of cytogenetic tumor research. Interest has been focused upon technically comparatively easy materials such as neoplastic hematological diseases, malignant exudates, and cell lines. Keeping in mind the fact that available data have been biased because of technical shortcomings, there exist several types or groups of neoplasms or neoplastic conditions that have been investigated to such an extent as to enable interpretations and assessments. The disorders chosen for this purpose are meningiomas and some myeloproliferative and lymphoproliferative diseases. In several disorders, the gonosomes comprise the chromosome types, which are possibly also involved in the nonrandom superimposed deviations. This is of interest because studies of the occurrence of Barr bodies and Y-bodies in tumor cells of mammary carcinomas, bladder carcinomas, and lung carcinomas and a variety of other neoplasms have indicated a fairly common loss of sex chromosomes in these cases. The reasons for this possible common loss of sex chromosomes in various neoplasms are at the current time completely obscure.
The karyotypes of cells from 10 Burkitt lymphoma (BL) biopsies, eight cell lines established from BL and nine cell lines from non-BL sources were studied by chromosome banding techniques. With the exception of the BL-derived cell lines BJAB, GC-BJAB, Maku and U-8691 all biopsies and lines of Burkitt origin contained an extra band at the distal region of the long arm of one chromosome 14. An extra band on chromosome 14 was also found in cells of one non-BL biopsy, in cells from a lymphosarcoma-derived cell line and in a long-established cell line derived from the pleural exudate of a patient with Hodgkin's disease. A distal region at the long arm of one chromosome 8 was missing in all metaphase figures of good technical quality in the same material. The size, morphology and stain-ability of the missing region corresponded fairly well to the extra region at chromosome 14. We therefore suggest that the chromosome 14 marker represents a translocation between chromosomes 8 and 14,t (8q-; 14q+). The translocation was present neither in lymphocytes of the peripheral blood of five Burkitt patients nor in five lymphoblastoid cell lines of non-BL origin. Trisomy 7 was found in two of the 10 BL biopsies, in two BL-derived cell lines, in one non-BL biopsy, in two lymphosarcoma-derived cell lines and in one cell line derived from a patient with Hodgkin's disease.
A nonrandom pattern of chromosomal abnormalities occurs in bone marrow cells obtained from patients with hematologic disorders who have an abnormal karyotype involving a C group chromosome. An additional number 8 chromosome is the most common abnormality, found in more than one-half of the patients studies. An additional number 9 chromosome and the loss of all or part of a number 7 are abnormalities that occur more often than might be expected by chance. It is proposed that specific human chromosomal abnormalities may be related to different specific etiologic agents.
The present survey comprises 856 cases of human neoplasms in which 1 or more chromosome aberrations have been identified by banding techniques. Of these, 704 cases had been published by the end of 1977, and 152 cases have not yet been published. The material is divided into 15 groups of malignant or premalignant disorders belonging to myeloproliferative disorders, lymphoproliferative disorders and solid tumors with 571, 160 and 125 cases, respectively. Most of the material (774 cases) is presented in tables, in which the chromosomes involved in aberrations are listed separately for each country. The main conclusion of the survey is that the different chromosome types are involved in aberrations to a strikingly different extent. The selective involvement, combined from all groups of diseases, clusters to 12 of the 24 human chromosome types, and 8 chromosome types are involved in 3–9 diseases each. There are suggestive indications that geographic differences in chromosome aberrations may exist. Agent dependence and tissue specificity of chromosome aberrations in neoplastic cells are discussed tentatively, and a working hypothesis is outlined concerning the possible role of chromosomal aberrations in oncogenesis.
Three patients with myeloproliferative disorders showed a similar chromosome abnormality, accompanied by other abnormalities that were different in each case. Marrow cells from all three patients were trisomic either for the entire chromosome 1 or for its long arm. Patient 1 had a brief period of anemia and thrombocytopenia which preceded a terminal acute leukemia; Patient 2 had polycythemia vera (P.V.) that terminated in acute leukemia; and Patient 3 has P.V. The detection of an abnormal karyotype in Patients 1 and 2 was an important factor in establishing the diagnosis of acute leukemia. Preliminary evidence supports the suggestion that some chromosomal changes are nonrandom in myeloproliferative diseases. Nonrandom abnormalities involving the same chromosome have been observed in several human neoplastic disorders.
KNOWLEDGE of the detailed pattern of fluorescence of the normal human karyotype, showing more than 200 bands per haploid chromosome set1, has enabled us to recognize, both in biopsies and in cell cultures from several Burkitt lymphomas, an extra band in one homologue of D group chromosome pair No. 14. The deviation was seen in all analysable cells of five out of six tumour biopsies and of seven out of nine tumour cell lines examined, representing twelve different tumours from nine male and three female patients. In three tumours both biopsies and cultures were examined, and it was found that all gave results consistent in the two types of samples. Thus, two of them had the marker band in both the biopsy and culture, the third revealed the marker band absent in both cases. The remaining tumours were investigated only in biopsies or only in culture. They were positive in eight cases, negative in one. Of the twelve tumours examined altogether, ten were positive and two negative.
Thirty-five cell lines were established from 47 Burkitt lymphoma biopsies. In different biopsies the modal cell size was found to vary and the tumor was often built up by larger cells if the patients received chemotherapy. In all cases the predominant cell size shifted towards larger values during cultivation. All cells in three biopsies from one patient and in the culture lines derived showed surface reactivity with anti-IgM and antikappa light chain reagents. This trait was maintained in all three lines for about 21 weeks but was lost thereafter. A fourth biopsy—taken after the patient had been treated with cytosine arabinoside—did not have this immunoglobulin reactivity. Chromosomal analysis revealed that one of the IgM reactive lines had 46 as stemline number and normal diploid karyotype, while the nonreactive line had 47 as stemline number with an extra C chromosome in addition to the normal male karyotype. One or two C chromosomes, corresponding in size with pair 10 were formed as markers with subterminal constriction on their long arms. The unusual cellular trait—IgM specificity—being present on both the biopsy and its derived cell line, indicates the representativeness of the culture line for the in-vivo tumor in this case.
The chromosomes of a lymphoblastic sarcoma were investigated in 2 biopsies from the malignant lymph nodes. Both biopsies had the same highly stable stemline karyotype, analyzed both by the classical orcein technique, including tritium autoradiography and measurements of all chromosomes of 10 cells, and by the new fluorescence technique. In relation to the normal human karyotype, the stemline had undergone changes in the proportions of the normal chromosome types and had acquired several marker chromosomes. With the aid of fluorescence technique some of the marker chromosomes were identified as parts of normal chromosomes, whereas others were not recognized. The latter included 3 medium-sized metacentrics with completely median centromere and the same fluorescence pattern in both arms. They were similar in every respect to marker chromosomes recently found in 4 other malignant lymphomas. The possible significance of these findings is discussed.
A FEW chromosome aberrations seem to recur with significant frequency
among the many aneuploid human lymphoblastoid lines established from
various sources. (For discussion and references see ref. 1.) These
include trisomy C, a marker chromosome (``mB'') larger than
the chromosomes of group 4/5 but with a similar arm ratio and an
acrocentric marker (``mD'') similar to an enlarged D group
chromosome.