Transition to tetraploidy in 1,25-dihydroxyvitamin D3-resistant HL60 cells is preceded by reduced growth factor dependence and constitutive up-regulation of Sp1 and AP-1 transcription factors.

Increased ploidy is an ominous event in the progression of human malignancies. It is usually associated with an increased growth rate of the neoplastic cells and a generally more autonomous and aggressive biological behavior. However, it has not been established whether the more rapid growth rate and growth factor independence are consequences of the polyploid, karyotypically increasingly aberrant nature of these cells or whether the accelerated, more autonomous growth contributes to polyploidization. In this study, we have examined a recently described (H. J. Wajchman et al., Exp. Cell Res., 224: 312-322, 1996) series of sublines of HL60 cells with increasing resistance to the monocytic differentiation-inducing steroid hormone 1,25-dihydroxyvitamin D3 (1,25D3) and found that growth factor independence, shown by reduced requirement for serum supplementation of the medium and the ability to grow at low seeding densities, precedes polyploidization of these cultures. The growth factor independence was found to be accompanied by constitutive changes in the DNA binding pattern of the ubiquitous transcription factor Sp1, characteristic of an exposure to 1,25D3. Similar changes in the pattern of AP-1 binding were also observed in the 1,25D3-resistant HL60 sublines, but the intensity of the DNA binding by AP-1 was increased only in sublines with resistance to 1,25D3 but still near-diploid. The data suggest that the culture of HL60 cells in the presence of 1,25D3 results in constitutive up-regulation of growth-related machinery that reduces the need for growth factors and cytokines and demonstrate that this increased growth potential precedes polyploidization of the culture populations.

Marrow fibrosis associated with a Philadelphia chromosome.

Three patients had marked marrow fibrosis and an apparent Philadelphia (Ph) chromosome. Hematologic, cytogenetic, and molecular studies demonstrated the heterogeneity of such cases, including the first example of clinically typical myelofibrosis (MF) associated with a bcr gene rearrangement characteristic of chronic myelogenous leukemia (CML).

Ph-negative T cells in a patient with chronic myelogenous leukemia for twenty-eight years.

The frequency of metaphases without a Philadelphia chromosome was determined in mitogen-stimulated cultures of peripheral blood mononuclear cells (PBMC) and purified T lymphocytes (93% CD2-positive) from a patient with chronic myelogenous leukemia (CML) for 28 years. The PBMC cultures contained few Ph-negative cells (8%), but they constituted 92% of the metaphases in T cell cultures, indicating few if any Ph-positive T cells in the patient's circulation. The results demonstrate that T cells derived from the leukemic clone may fail to replace the non-neoplastic population even when CML arises in childhood and the patient survives for many years. This raises questions concerning the normal role of the bone marrow as a source of T cells after infancy, and also whether Ph-positive lymphocytes may be at a disadvantage for growth.

Molecular cloning and characterization of a cDNA for human granulocyte colony-stimulating factor (G-CSF) from a glioblastoma multiforme cell line and localization of the G-CSF gene to chromosome band 17q21.

The conditioned media (CM) of the glioblastoma multiforme cell line, U87 MG, contains abundant granulocyte colony-stimulating factor (G-CSF) activity (Tweardy et al., 1987). An oligonucleotide encoding the amino acids -11 to -4 of G-CSF detected a single abundant G-CSF mRNA of 1.6 kilobases (Kb) produced by U87 MG cells. Screening of a U87 MG cDNA library with the oligonucleotide identified cDNA clones of 1.3-1.4 Kb. Sequencing of one clone (pG-CSF6) confirmed that it encoded G-CSF and was derived from G-CSFb mRNA encoding a protein with a three amino acid deletion at positions 36-38. Only a single base substitution was observed at the third position of the codon for leu 152 indicating that G-CSF is highly conserved in cells of widely different origin. Somatic cell hybridization studies and chromosomal in situ hybridization localized the G-CSF gene to the long arm of chromosome 17 in band 17q21, proximal to the 17q breakpoint characteristic of acute promyelocytic leukemia.

Chromosome studies in preleukemic states. V. Prognostic significance of single versus multiple abnormalities.

The prognostic value of marrow chromosome findings was examined in 242 patients with preleukemic myelodysplastic syndromes (MDS) or myeloproliferative disorders (MPD), with emphasis on the significance of single versus multiple karyotypic changes. In both groups, the results showed that patients with multiple chromosome abnormalities in a marrow clone had a very high probability of early death, from progression to leukemia or from other complications of hematopoietic dysfunction. Conversely, in patients with a hemic clone having only one karyotypic alteration (involving a single chromosome or single translocation), survival over 2 years was only slightly reduced as compared to those without chromosome abnormality. The only single karyotypic alteration perhaps associated with a markedly shortened survival was monosomy 7. These findings suggest that the conclusions of previous studies concerning the grave consequences of chromosome alterations in preleukemia largely reflect the clinical significance of clones with multiple cytogenetic changes. Prior knowledge of the karyotypic status of preleukemic patients should be helpful in evaluating current attempts to find effective treatment for these difficult disorders.

The most common chromosome change in 86 chronic B cell or T cell tumors: a 14q32 translocation.

Among 46 patients with chronic lymphocytic leukemia (CLL) (40 B cell, 6 T cell) and 40 patients with cutaneous T cell lymphoma (CTCL), a chromosomally abnormal neoplastic clone was identified in 43 cases. A translocation involving 14q32 was present in nine cases (five B-CLL, two T-CLL, two CTCL). The donor chromosomal site was 11q13 in four patients and 1q12, 4q25-27, 17q21-22, 18q21, and 22q11 in one case each. The next most frequent abnormalities were rearrangements involving 6q21-23 (four cases), and trisomy 12 (four cases, all B-CLL). In one CTCL patient, the t(11;14) translocation was present in one of three apparently unrelated T cell clones. Recent studies indicate that the selective advantage conferred by the 14q+ chromosome in B cell neoplasms appears to result from an oncogene being brought adjacent to a rearranged and transcriptionally active immunoglobulin heavy chain locus. The present findings suggest that similar mechanisms may operate in certain T cell neoplasms, although the activating gene is not necessarily the same.

Diagnostic and prognostic significance of Sézary cells in peripheral blood smears from patients with cutaneous T cell lymphoma.

Blood smears stained with Wright-Giemsa were obtained from 124 patients with pathologically confirmed cutaneous T cell lymphoma (CTCL), 70 patients with various other cutaneous disorders, and ten healthy adult volunteers. These were examined in a blinded fashion for atypical lymphocytes with cerebriform nuclei (CLs), which were characterized further according to cell diameter. CLs, comprising up to 15% of lymphocytes in smears, were observed in 20% of the patients with benign dermatitis. CLs, comprising up to 89% of lymphocytes in smears, were found in 22%, 30%, 50%, and 96% of patients with patch, plaque, tumor, and erythrodermic CTCL, respectively. Large-diameter CLs (15 to 20 micron) were observed only in smears from patients with CTCL. Total CL counts above 15 per 100 lymphocytes and/or the presence of large CLs occurred in 33 of 49 (67%) patients with erythrodermic disease and in only two patients with other skin manifestations. Blood smears obtained at the time of cytogenetic studies indicated that a total CL count above 15% was the smear criterion that correlated best with the demonstration of a chromosomally abnormal malignant clone in the blood. The presence of large CLs per se, although also predictive of a malignant clone, was less useful. Multivariate survival analysis showed that the duration of disease before the blood smear and the proportion of large CLs within the total CL population were the covariates that correlated most significantly with survival. We speculate that the reduced survival of patients with increased proportions of large CLs in smears reflects the presence of polyploid malignant lymphocytes in the blood.

Chromosome rearrangements in oncogenesis.

Chromosomal studies have earlier provided evidence for the clonal nature of most neoplasms, and for the role of sequential genetic change in tumour progression. Now, in combination with molecular techniques, they are indicating how the function of specific genes (oncogenes) can be significantly altered by chromosomal translocations or by gene amplification, contributing to carcinogenesis.

Karyotypic differences between primary cultures and cell lines from tumors with the human T-cell leukemia virus.

Chromosome abnormalities were studied in primary cultures and in established T-cell lines from patients with human T-cell leukemia virus (HTLV)-positive leukemia or lymphoma. The present findings, and data from other laboratories, indicated that primary cultures of the HTLV-positive neoplastic cells nearly always showed a chromosomally abnormal clone, whereas most established cell lines had an apparently normal karyotype. These differences included circumstances in which the same blood specimen was used for both types of culture or in which separate specimens were obtained within a short time span. These observations indicated that many cell lines from HTLV-positive leukemia or lymphoma may be derived from nonneoplastic T-cells that were transformed in vitro by the leukemia virus; human T-cells newly infected with HTLV were suggested to have an in vitro growth advantage over the HTLV-infected tumor cells.

Clonal characteristics of cutaneous T cell lymphomas: cytogenetic evidence from blood, lymph nodes, and skin.

Chromosome studies were done on mitogen-stimulated lymphocytes from one or more tissues (blood, lymph nodes, skin lesions) in 15 patients with mycosis fungoides or Sézary syndrome. A cytogenetically abnormal clone was found in 10 individuals, including 6 with data from several tissues. In 4 cases the same aberrant clone was identified in a skin lesion as well as in blood and/or lymph node. The abnormal chromosome patterns ranged from hypodiploid to hypertetraploid, and there was no evidence of unrelated karyotypically-altered lines at different sites. A 6q- chromosome, previously reported in acute lymphocytic leukemia, was found in 2 patients. The results support the concept that cutaneous T cell lymphomas (CTCL) are clonal disorders, presumably unifocal in origin, with the skin lesions populated by cells from the same neoplastic clone that involves lymph nodes and blood.

Cytogenetics of acute and chronic myelofibrosis.

In myelofibrosis, acute or chronic, as well as in other myeloproliferative disorders which carry an increased risk of developing leukemia, a clone of hemic cells with a chromosome abnormality is a relatively common occurrence. To date, however, the presence or absence of a cytogenetic alteration has not been of prognostic value with respect to subsequent clinical course. No particular karyotypic change is specific for myelofibrosis, but many of the same non-random abnormalities occur as in other leukemic and preleukemic states. Both cytogenetic and isoenzyme data indicate that the fibrous tissue in the marrow is not part of the myeloproliferative clone.

Proliferation in vivo of T lymphocytes reactive to histocompatibility alloantigens: a correction.

Rat lymphocytes in mixed cultures can reutilize tritiated thymidine from labeled granulocytes. Shortly after thymidine injections in vivo, major effects on the frequency of labeled lymphocyte mitoses in peripheral blood cultures are introduced by 10-20% polymorph contamination, even though transfer of label via supernates is not demonstrable. Cold thymidine in the cultures prevents reutilization, and has permitted reevaluation of several previous conclusions concerning the life history of lymphocytes reactive to major histocompatibility alloantigens (HARC). Rather than being predominantly recently divided cells, HARC do not appear to have an age distribution, in blood or lymph, significantly different from the general recirculating lymphocyte population. However, the ability of immunization across strong allogeneic differences to increase markedly the proportion of young HARC among the specifically responsive population has been confirmed.