Gene expression profiling of fibroblasts resistant toward oncogene-mediated transformation reveals preferential transcription of negative growth regulators.

The signal-transducing Ras proteins are important driving forces of diverse cellular processes such as proliferation, neoplastic transformation, differentiation and growth inhibition. As a step toward understanding the complex mechanisms underlying cellular responses, gene expression patterns were examined in two phenotypically normal fibroblast lines which differ in their sensitivity toward oncogene-mediated transformation. Suppression subtractive hybridization (SSH) was used to establish a subtracted cDNA library specific for the REF52 cell line which, like normal diploid fibroblasts, is refractory toward neoplastic transformation induced by mutated HRAS oncogenes. In contrast, rat 208F control cells can be efficiently transformed by HRAS. The nucleotide sequence of 549 subtracted cDNA clones ('REF52 minus 208F') was determined. We identified 93 preferentially expressed gene fragments in resistant REF52 cells as compared to 208F cells. Seventeen of the 52 known genes (32.6%) are capable of inhibiting cell proliferation or of adversely affecting oncogenic signal transduction pathways. These results suggest that the anti-oncogenic properties of resistant REF52 cells are determined by multiple negative growth regulators. The preneoplastic state expressed in 208F cells is characterized by impairment of unexpectedly redundant control mechanisms. Our results also demonstrate that SSH is a powerful method for identifying specific transcriptional patterns in closely related cell types.

Regulation of 15-lipoxygenase expression by cytokines.

The arachidonate 15-lipoxygenase is induced in peripheral human monocytes by culturing the cells for 3 days in the presence of interleukin 4 (IL-4) in concentrations as low as 40 pM. Linoleic acid is oxygenated by IL-4 treated monocytes to 13(S)-hydroxy-9Z, 11E-octadecadienoic acid [13(S)-HODE] with a specific activity of about 2 nmoles 13(S)-HODE/10(6) cells min. A screening of various permanent cell lines expressing the IL-4 receptor indicated that all monocyte/macrophage lines tested did not exhibit the effect of LOX induction. However, IL-4 treatment of the lung carcinoma cell line CCC 185 and of the colon carcinoma cell line HTB 38 induces the 15-LOX as shown by activity assay and immunohistochemistry. The IL-4 mutant Y124D which has been characterized as specific IL-4 receptor antagonist in human T-cells does not induce the 15-LOX but appears to act as competitive inhibitor for the induction. Subcellular fractionation of IL-4 treated monocytes indicated a cytosolic and a membrane bound enzyme pool. The intracellular action of the LOX leads to a specific oxygenation of the membrane phospholipids which is drastically increased after damage to the cells. The possible biological role of the 15-LOX for monocyte metabolism is discussed.

Specificity of PCR-based clonality analysis of immunoglobulin heavy chain gene rearrangements for the detection of bone marrow involvement by low-grade B-cell lymphomas.

A study was performed to investigate the utility of polymerase chain reaction (PCR)-based analysis of immunoglobulin heavy chain (IgH) gene rearrangements for the diagnosis of low-grade malignant B-cell lymphomas on formalin-fixed, EDTA-decalcified, and paraffin-embedded bone marrow trephine biopsies. On amplifying two DNA samples per biopsy, no reproducible monoclonal PCR result was found in 32 patients with reactive lymphoid hyperplasias. In contrast, 5/14 patients with known low-grade B-cell lymphomas, but histomorphologically and immunohistochemically lymphoma-free bone marrow, showed a reproducible monoclonal IgH gene rearrangement. In three of these cases, sequence analysis revealed completely different amplification products on comparing bone marrow and lymph node infiltrations, while in the other two cases the products were identical. In one of the latter biopsies, an unequivocal lymphoma infiltrate was found after step sectioning of the biopsy, while the other case remained lymphoma-free according to conventional criteria. A third group of three patients with known lymphomas and bone marrow findings that were suggestive but not diagnostic of bone marrow involvement showed monoclonal PCR results in all three cases, with identical sequences in bone marrow and extramedullary lymphoma infiltrates. These data suggest that a reproducible monoclonal IgH gene rearrangement is highly specific for the presence of malignant B-cells in bone marrow. In staging procedures for low-grade B-cell lymphomas, PCR yields no additional information in cases that are morphologically and immunohistochemically lymphoma-free after evaluation of representative sections. PCR may be useful in equivocal cases, provided that IgH gene rearrangements of extramedullary lymphoma and bone marrow are sequenced and compared.

Membrane translocation of 15-lipoxygenase in hematopoietic cells is calcium-dependent and activates the oxygenase activity of the enzyme.

Mammalian 15-lipoxygenases, which have been implicated in the differentiation of hematopoietic cells are commonly regarded as cytosolic enzymes. Studying the interaction of the purified rabbit reticulocyte 15-lipoxygenase with various types of biomembranes, we found that the enzyme binds to biomembranes when calcium is present in the incubation mixture. Under these conditions, an oxidation of the membrane lipids was observed. The membrane binding was reversible and led to an increase in the fatty acid oxygenase activity of the enzyme. To find out whether such a membrane binding also occurs in vivo, we investigated the intracellular localization of the enzyme in stimulated and resting hematopoietic cells by immunoelectron microscopy, cell fractionation studies and activity assays. In rabbit reticulocytes, the 15-lipoxygenase was localized in the cytosol, but also bound to intracellular membranes. This membrane binding was also reversible and the detection of specific lipoxygenase products in the membrane lipids indicated the in vivo activity of the enzyme on endogenous substrates. Immunoelectron microscopy showed that in interleukin-4 -treated monocytes, the 15-lipoxygenase was localized in the cytosol, but also at the inner side of the plasma membrane and at the cytosolic side of intracellular vesicles. Here again, cell fractionation studies confirmed the in vivo membrane binding of the enzyme. In human eosinophils, which constitutively express the 15-lipoxygenase, the membrane bound share of the enzyme was augmented when the cells were stimulated with calcium ionophore. Only under these conditions, specific lipoxygenase products were detected in the membrane lipids. These data suggest that in hematopoietic cells the cytosolic 15-lipoxygenase translocates reversibly to the cellular membranes. This translocation, which increases the fatty acid oxygenase activity of the enzyme, is calcium-dependent, but may not require a special docking protein.

An unusual case of leukemic non-Hodgkin's lymphoma with blastic transformation.

We report on a patient who was diagnosed as having B-cell chronic lymphocytic leukemia (CLL) with atypical morphology. Flow cytometry disclosed CD5, CD19, and CD23 positivity, an immunophenotype seen mostly in B-CLL. Histology of the spleen and bone marrow suggested a diagnosis of small lymphocytic lymphoma. Upon blastic transformation, only 3 years after the diagnosis had been made, unusual clinical and laboratory features emerged. Lymphoid blasts appeared in the peripheral blood, and the patient developed nodular infiltrates consisting of these blasts at recent venous puncture sites. The patient did not respond to chemotherapy and died. The lymphoid blasts in the peripheral blood were CD5-, CD19+, and CD23+ and harbored t(11;14) (q13;q32) and t(11;21)(p11;q21) translocations. To account for the possibility of two independent lymphoid malignancies, molecular genetic analyses were performed on samples from the spleen, bone marrow and a lymph node with the large-cell lymphoma, which showed identical clones in these tissues. This unusual case supports the idea that in leukemic non-Hodgkin's lymphoma, in addition to morphology, an accurate diagnostic workup requires immunophenotypic, cytogenetic, and molecular studies.

Regulation of 15-lipoxygenase expression in lung epithelial cells by interleukin-4.

We have studied the expression of the 15-lipoxygenase gene in various permanent mammalian cell lines in response to interleukins-4 and -13, and found that none of the cell lines tested expressed 5-, 12- or 15-lipoxygenase when cultured under standard conditions. However, when the lung carcinoma cell line A549 was maintained in the presence of either interleukin for 24 h or more, we observed a major induction of 15-lipoxygenase, as indicated by quantification of 15-lipoxygenase mRNA, by immunohistochemistry, by immunoblot analysis and by enzyme activity assays. This effect was 15-lipoxygenases-specific, since expression of 5- and 12-lipoxygenases remained undetectable. The time course of interleukin-4 treatment indicated maximal accumulation of both 15-lipoxygenase mRNA and functional protein after 48 h. Binding studies revealed that A549 cells express about 2100 high-affinity interleukin-4 binding sites per cell. The interleukin-4 mutant Y124D, which is capable of binding to the interleukin-4 receptor but is unable to trigger receptor activation, counteracted the effect of the wild-type cytokine. Other cell lines, including several epithelial cells and various monocytic cell lines expressing comparable numbers of interleukin-4 receptors, did not express 15-lipoxygenase when stimulated with interleukin-4. These data indicate that A549 cells selectively express 15-lipoxygenase when stimulated with interleukins-4 and -13. The activation of the interleukin-4/13 receptor(s) appears to be mandatory, but not sufficient, for 15-lipoxygenase gene expression.