Human mixed leukocyte culture: identification of the proliferating lymphocyte subpopulation by sex chromosome markers.

The nature of the cells which proliferate in response to allogeneic histoincompatible lymphocytes in human mixed leukocyte cultures (MLC) was investigated. Direct information on the differentiation of proliferating cells was obtained in an in vitro chimeric system with T- and B-cell sex chromosome markers. Highly purified T and B cells were prepared from the peripheral blood of three pairs of HL-A genotype identical, MLC-negative siblings of opposite sex by E-rosette sedimentation techniques. Recombined T and B cells of the siblings were stimulated with allogeneic cells, and karyotype analysis of proliferating cells performed. 96% of the proliferating cells were found to carry the sex chromosome marker of the respective T-cell population. These results indicate that proliferation in human MLCs is a feature of T cells.

Isopentenyladenosine stimulates and inhibits mitosis of human lymphocytes treated with phytohemagglutinin.

The plant cytokinin isopentenyladenosine, a component of yeast and mammalian transfer ribonucleic acid, is both a potent inhibitor and stimulator of DNA synthesis, transformation, and mitosis of the phytohemagglutinin-stimulated lymphocyte. The stage of the cell cycle and the concentrations used are critical for these effects. The addition of isopentenyladenosine within the first 12 hours after phytohemagglutinin at a concentration above 10(-6) molar results in inhibition, while lower concentrations (between 10(-7) and 10(-6) molar), added at 24 hours or later, have a stimulatory effect.

Hemopoietic stem cells in human peripheral blood.

A population of lymphocytes, separable from the great majority by virtue of their larger size and their failure to exhibit the rosetting characteristics of thymus-dependent lymphocytes and bursa-equivalent cells, possess true pluripotentiality. On culture in vivo they proliferate and differentiate into erythrocytic, granulocytic, and megakaryocytic progeny. This may be the first clear demonstration of the primitive progenitor blood cell in man.

Epithelial origin of polyoma salivary tumors in mice: evidence based on chromosome-marked cells.

Trypsin dissection of epithelium from mesenchyme of salivary gland rudiments allows reassembly of glands having either epithelium or mesenchyme selectively marked by T(6) chromosomes. Virus-induced tumors in such glands invariably bear the karyotype of the epithelial component. The method solves a specific example from a group of classical problems concerning epithelial as opposed to mesenchymal origin of neoplasms.

Abnormalities in structure and expression of the human retinoblastoma gene in SCLC.

Small cell lung cancer (SCLC) has been associated with loss of heterozygosity at several distinct genetic loci including chromosomes 3p, 13q, and 17p. To determine whether the retinoblastoma gene (Rb) localized at 13q14, might be the target of recessive mutations in lung cancer, eight primary SCLC tumors and 50 cell lines representing all major histologic types of lung cancer were examined with the Rb complementary DNA probe. Structural abnormalities within the Rb gene were observed in 1/8 (13%) primary SCLC tumors, 4/22 (18%) SCLC lines, and 1/4 (25%) pulmonary carcinoid lines (comparable to the 20 to 40% observed in retinoblastoma), but were not detected in other major types of lung cancer. Rb messenger RNA expression was absent in 60% of the SCLC lines and 75% of pulmonary carcinoid lines, including all samples with DNA abnormalities. In contrast, Rb transcripts were found in 90% of non-SCLC lung cancer lines and in normal human lung. The finding of abnormalities of the Rb gene in SCLC and pulmonary carcinoids (both neuroendocrine tumors) suggests that this gene may be involved in the pathogenesis of a common adult malignancy.

Synthesis of kappa light chains by cell lines containing an 8;22 chromosomal translocation derived from a male homosexual with Burkitt's lymphoma.

Three cell lines were derived from a homosexual patient with probable acquired immunodeficiency syndrome and Burkitt's lymphoma. The cell lines produce an unusual strain of Epstein-Barr virus which will both transform cord blood lymphocytes and induce early antigens in Raji cells. Translocations between chromosomes 8 and 22 have occurred in all three lines, but the cells synthesize immunoglobulin M with light chains of the kappa type, in contrast to the usual concordance between a translocation involving chromosome 22 and lambda chain synthesis. Both kappa genes and one lambda gene are rearranged. These findings indicate either that translocation may occur as a separate event from immunoglobulin gene rearrangement or that the proposed hierarchical sequence of immunoglobulin gene rearrangements is not always adhered to. The data also imply that in cells containing a translocation between the long arm of chromosome 8 and a chromosome bearing an immunoglobulin gene, alteration of cellular myc expression may occur regardless of the immunoglobulin gene that is expressed.

Specific chromosome defect associated with human small-cell lung cancer; deletion 3p(14-23).

A specific, acquired chromosomal abnormality (deletion 3p) has been found in at least one chromosome 3 in 100 percent of the metaphases in 12 of 12 cell lines cultured from human small-cell lung cancer tissue and in 2-day tumor culture specimens from three patients. Analysis of the shortest region of overlap shows the deletion to be 3p(14-23). This specific change was not seen in five of five lung cancer cell lines other than small-cell lung cancer or in two lymphoblastoid lines cultured from cells of small-cell lung cancer patients whose tumors had the 3p deletion.

DNA content analysis by flow cytometry and cytogenetic analysis in mycosis fungoides and Sézary syndrome.

Flow cytometric (FCM) analysis of DNA content was performed on 82 lymph node and peripheral blood specimens from 46 patients with mycosis fungoides and the Sézary syndrome. Overall, 32 of the 46 patients (70%) had aneuploidy detected by FCM. Aneuploidy was present in 63% of the patients at the time of diagnosis before systemic therapy. In these patients, aneuploidy was frequently detected in blood and lymph node specimens scored as negative by cytology and histology, suggesting that unsuspected extracutaneous dissemination is present in many patients at the time of diagnosis. Direct comparison with Giemsa-banded cytogenetic studies showed an excellent correlation of FCM results and cytogenetic chromosome number. However, FCM frequently detected a larger fraction of aneuploid cells, and mitogen-stimulation studies suggest this is the result of preferential stimulation of normal lymphocytes by phytohemagglutinin. Thus, mitogens with a preference for malignant T cells, such as staphylococcal protein A, should be used for cytogenetic analysis of malignant T-cell disorders. At diagnosis, some histologically positive specimens contained only diploid cells by FCM and cytogenetic analysis. These patients had a more indolent clinical course than patients with aneuploidy. Aneuploidy was detected by FCM as either wide G(1) or as discrete aneuploid peaks. The presence of aneuploidy at any time in the clinical course implied a poor prognosis. Discrete hyperdiploid peaks were associated with large cell histology, early relapse, and aggressive clinical course. The development of hyperdiploidy at relapse was documented in four patients and was associated with a transition to large cell histology and a poor prognosis. Similar studies may elucidate differences in natural history and mechanism for transition in histology in other lymphomas and solid tumors.

Absence of hereditary p53 mutations in 10 familial leukemia pedigrees.

Germline p53 mutations have been identified in the Li-Fraumeni syndrome but the role of such mutations in familial leukemia is not established. The p53 gene was examined by single-strand conformation polymorphism analysis of exons 4-8 in 10 families with multiple members affected with leukemia. The diagnoses included acute and chronic leukemias and Hodgkin's disease. Identified in two families were p53 mutations that were nonhereditary. These included a 2-bp deletion in exon 6 found in the lymphoblast DNA of one child whose sibling, cousin, and several adult relatives had acute leukemia. The other nonhereditary p53 mutation was a transition at codon 248 (CGG to CAG, arginine to glutamine) found in the lymphoblasts of a patient with a preleukemic syndrome and acute lymphoblastic leukemia (ALL) whose brother is a long-term survivor of ALL. Thus, p53 mutations were found to occur in two families but both were nonhereditary. Moreover, in the remaining eight families no p53 mutation was identified in the regions of p53 where most mutations have been found in other cancers. Although p53 mutations sometimes may be present, they do not appear to be a primary event responsible for hereditary susceptibility to familial leukemia. This study suggests involvement of other genes or mechanisms.

Hereditary and acquired p53 gene mutations in childhood acute lymphoblastic leukemia.

The p53 gene was examined in primary lymphoblasts of 25 pediatric patients with acute lymphoblastic leukemia by the RNase protection assay and by single strand conformation polymorphism analysis in 23 of 25 cases. p53 mutations were found to occur, but at a low frequency (4 of 25). While all four mutations were identified by single strand conformation polymorphism, the comparative sensitivity of RNase protection was 50% (2 of 4). Heterozygosity was retained at mutated codons in 3 of 4 cases. One pedigree was consistent with the Li-Fraumeni syndrome, and bone marrow from both diagnosis and remission indicated a germline G to T transversion at codon 272 (valine to leucine). Although members of another family were affected with leukemia, a 2-bp deletion in exon 6 was nonhereditary. The other two nonhereditary p53 mutations included a T to G transversion at codon 270 (phenylalanine to cysteine) and a G to C transversion at codon 248 (arginine to proline). These data support the role of both hereditary and acquired p53 mutations in the pathogenesis and/or progression of some cases of childhood acute lymphoblastic leukemia.

Structure and localization of genes encoding aberrant and normal epidermal growth factor receptor RNAs from A431 human carcinoma cells.

A431 cells have an amplification of the epidermal growth factor (EGF) receptor gene, the cellular homolog of the v-erb B oncogene, and overproduce an aberrant 2.9-kilobase RNA that encodes a portion of the EGF receptor. A cDNA (pE15) for the aberrant RNA was cloned, sequenced, and used to analyze genomic DNA blots from A431 and normal cells. These data indicate that the aberrant RNA is created by a gene rearrangement within chromosome 7, resulting in a fusion of the 5' portion of the EGF receptor gene to an unidentified region of genomic DNA. The unidentified sequences are amplified to about the same degree (20- to 30-fold) as the EGF receptor sequences. In situ hybridization to chromosomes from normal cells and A431 cells show that both the EGF receptor gene and the unidentified DNA are localized to the p14-p12 region of chromosome 7. By using cDNA fragments to probe DNA blots from mouse-A431 somatic cell hybrids, the rearranged receptor gene was shown to be associated with translocation chromosome M4.

Inverted repeats as genetic elements for promoting DNA inverted duplication: implications in gene amplification.

Inverted repeats are important genetic elements for genome instability. In the current study we have investigated the role of inverted repeats in a DNA rearrangement reaction using a linear DNA substrate. We show that linear DNA substrates with terminal inverted repeats can efficiently transform Escherichia coli. The transformation products contain circular inverted dimers in which the DNA sequences between terminal inverted repeats are duplicated. In contrast to the recombination/rearrangement product of circular DNA substrates, which is exclusively one particular form of the inverted dimer, the rearrangement products of the linear DNA substrate consist of two isomeric forms of the inverted dimer. Escherichia coli mutants defective in RecBCD exhibit much reduced transformation efficiency, suggesting a role for RecBCD in the protection rather than destruction of these linear DNA substrates. These results suggest a model in which inverted repeats near the ends of a double-strand break can be processed by a helicase/exonuclease to form hairpin caps. Processing of hairpin capped DNA intermediates can then yield inverted duplications. Linear DNA substrates containing terminal inverted repeats can also be converted into inverted dimers in COS cells, suggesting conservation of this type of genome instability from bacteria to mammalian cells.

Suppression of gene amplification and chromosomal DNA integration by the DNA mismatch repair system.

Mismatch repair (MMR)-deficient cells are shown to produce >15-fold more methotrexate-resistant colonies than MMR normal cells. The increased resistance to methotrexate is primarily due to gene amplification since all the resistant clones contain double-minute chromosomes and increased copy numbers of the DHFR gene. In addition, integration of linearized or retroviral DNAs into chromosomes is also significantly elevated in MMR-deficient cells. These results suggest that in addition to microsatellite instability and homeologous recombination, MMR is also involved in suppression of other genome instabilities such as gene amplification and chromosomal DNA integration.

Serial cytogenetic studies of nonendemic Burkitt's lymphoma cell lines.

Cytogenetic study of nonendemic Burkitt's lymphoma (BL) cell lines showed a translocation of chromosomes #8 and #14, t(8;14), present in 16 of the 18 lines examined. Serial cytogenetic studies of nine of these lines showed the t(8;14) to be stable and present in all cells examined. Although many other chromosome aberrations were present, they did not demonstrate the stability or the pervasiveness of the t(8;14). The significance of these results and previously reported cytogenetic studies on the etiology of BL was discussed.

Mutant lines of guinea pig L2C leukemia. II. Comparative cytogenetic studies and banding analyses of normal and leukemic karyotypes.

Chromosomes of normal guinea pig (strain 2) cells and of cell lines dervied from a spontaneously arising leukemia were analyzed in detail. All cell lines studied, LG-L2C, GH-L2-C, BZ-LC, and EN-L2C, contained one M1 marker and two X chromosomes, in addition to other chromosome abnormalities specific for each cell line. The presence of the M1 marker and the two X chromosomes confirmed that all of these leukemic cell lines are derived from one ancestral line. Comparison of the chromosome markers and immunologic characteristics of these lines revealed that possibly the gene involved in the determination of C3 receptor sites is located on the terminal portion of the long arm of chromosome No. 2, but no other correlations could be made.

Direct cytogenetic studies by needle aspiration of Burkitt's lymphoma in Ghana, West Africa.

With the use of a needle aspiration technique for obtaining tumor material, the direct karyotype of Burkitt's lymphoma cells was examined from 15 Ghanaian patients. The 14q+ chromosome (in addition to other chromosomal abnormalities) was present in 8 (57%) of the 14 patients; 6 of these 8 patients had t(8;14) (q24;q32). One of the remaining 2 patients had a #8 chromosome deleted, whereas the second had 2 normal-appearing #8 chromosomes. Other numerical and structural abnormalities were frequent. No correlation between type or frequency of abnormality and clinical status or survival was found. Within cells of the same tumor aspirate, the karyotype abnormalities were generally similar. In 3 patients, karyotypes from two separate tumor sites (abdomen-bone marrow, 2 cases; right maxilla-left mandible, 1 case) showed identical markers. The same marker was seen in specimens from the initial and second relapse abdominal tumors of 1 patient. The similarity of chromosomal markers from two sites or in serial studies further supports the concept that African Burkitt's lymphoma is a disease of monoclonal origin.

Cytogenetic studies of human breast cancer lines: MCF-7 and derived variant sublines.

Cytogenetic studies of the human breast cancer cell line MCF-7 and the sublines derived from it demonstrated extensive aneuploidy with both numerical and structural abnormalities and a wide range of heteroploidy. Detailed G-banding analyses showed that loss of marker chromosomes was a rare event, while formation of additional structural abnormalities was very common in these long-term cultures. All chromosomes were involved in these abnormalities. Loss of a morphologically normal X-chromosome may be related to the loss of estrogen receptors in these cell lines. With the exception of one subline, all cell lines had a short homogeneously staining region (HSR) on chromosome 7. Although the parent MCF-7 line had tetrahydrofolate dehydrogenase levels within the normal range, a lengthened HSR has been found in MCF-7 lines that are resistant to methotrexate. This observation strongly favors the association of an increased level of tetrahydrofolate dehydrogenase with HSR. In conclusion, the inherent genetic instability in this group of related cell lines may explain the heterogeneity found in this tumor. Continuous chromosomal rearrangements and numerical changes may reflect an ongoing process of selection and adaptation in these cell lines established from a breast carcinoma and may be characteristic of the aggressiveness of this neoplasm.

Characterization of lymphoma-derived cell lines: comparison of cell lines positive and negative for Epstein-Barr virus nuclear antigen. I. Physical, cytogenetic, and growth characteristics.

Sixteen lymphoid cell lines were derived from patients with undifferentiated lymphoma of Burkitt's or non-Burkitt's type. They were obtained directly from tumor biopsies, from serous effusions, or from bone marrow. In 10 of the cell lines, the Epstein-Barr virus (EBV) nuclear antigen (EBNA) was undetectable; the remaining 6 lines were EBNA-positive (EB-pos). Of the 16 lines, 15 were aneuploid, with detectable chromosome "14q+ markers (11 had +8;14 translocations). These 15 lines, which included the EBNA-negative (EB-neg) lines, were believed to be of tumor cell origin. The remaining line consisted predominantly of diploid cells derived from normal lymphocytes, but some cells of tumor origin were present. Four EB-pos cell lines derived from EB-neg tumors had an aneuploid karyotype consistent with an origin from tumor cells (including no.8;14 translocation in two), which suggested that either tumor cells were infected with EBV in vitro or a tiny fraction of EB-pos tumor cells (or potential tumor cells) present in vivo gave rise to the predominant cell of the line. EB-neg B-cell lines and EB-pos cell lines established from undifferentiated lymphomas differed greatly. EB-neg lines had consistently smaller electronic mean cell volumes and narrow-angle light scatter than did EB-pos lines. This finding correlated with a lower nuclear:cytoplasmic ratio in EB-pos lines. EB-neg lines also had higher saturation cell densities than did EB-pos lines under standard culture conditions. The data indicate either that EBV influences the morphologic and physiologic characteristics of lymphoid cell lines or that EB-neg B-cell lines and EB-pos cell lines are derived ultimately from different lymphocyte subpopulations or that both may apply.

Deletion of 3(p14p23) in secondary erythroleukemia arising in long-term survivors of small cell lung cancer.

Cytogenetic studies were done on the leukemia cells of two patients with small cell lung cancer (SCLC) who developed erythroleukemia (acute nonlymphocytic leukemia, French-American-British M6) after combined modality chemotherapy and radiotherapy for their lung cancer. Surprisingly, both erythroleukemias exhibited the del(3)(p14p23) predominantly found in SCLC. In four other patients who had secondary erythroleukemias associated with other cancers, no deletions of 3p were found. These findings could be accounted for by one of three possible mechanisms: (a) an inherited recessive gene (anti-oncogene or tumor suppressor gene) in this region of 3p was uncovered by the combined modality therapy, (b) an inherited predisposition to damage of both chromosomes at 3p14 leads to SCLC and erythroleukemia after exposure to carcinogens and/or chemotherapy-radiotherapy, or (c) the finding of lineage specificity for the 3p deletion with the presence of the 3p deletion in SCLC and erythroleukemia suggests a common bone marrow precursor.