Four exons encode a 93-base-pair insert in three neural cell adhesion molecule mRNAs specific for chicken heart and skeletal muscle.

The neural cell adhesion molecule (N-CAM) is detected in chicken brain as three polypeptides of 180 kDa, 140 kDa, and 120 kDa that arise from a single gene by alternative splicing. Heart tissue, however, contains components of 150 kDa, 140 kDa, and 130 kDa; neither the differences in molecular mass among these components nor the difference between neural and cardiac N-CAM could be accounted for by variations in glycosylation alone. A cDNA clone isolated from an embryonic chicken heart library, [lambda N101B, 1.8 kilobases (kb)] contained a 93-base-pair (bp) insert not found in neural N-CAM cDNAs. In the N-CAM gene this sequence mapped within a large region between exons 12 and 13 and was derived from four exons (12A-D) of 15, 33, 42, and 3 bp. Exons 12C and 12D together coded for 15 amino acids very similar to the second half of the muscle-specific insert (MSD1) found in N-CAM cDNA from human muscle cell cultures [Dickson, G., Gower, H. J., Barton, C. H., Prentice, H. M., Elsom, V. L., Moore, S. E., Cox, R. D., Quinn, C., Putt, W. & Walsh, F. S. (1987) Cell 50, 1119-1130]; the sequences of 12A and 12B, however, were much less similar to the corresponding region of the MSD1 sequence. Two oligonucleotides, one specific to exons 12A plus 12B and one specific to exon 12C both recognized mRNA species of 6.4 kb, 4.3 kb, and 3.0 kb in chicken cardiac and skeletal muscle and no mRNA species in smooth muscle or brain. The 3' end of clone lambda N101B contained a sequence coding for a potential phosphatidylinositol linkage signal as does the smallest form of brain N-CAM. In heart cell membranes only the 130-kDa N-CAM polypeptide was released by phospholipase C, suggesting that this form of N-CAM is encoded by clone lambda N101B. The other heart N-CAM species (150 kDa and 140 kDa) may be transmembrane forms that include the 12A-D (and possibly other) inserts. Tissue-specific forms of N-CAM can thus be formed by alternative use of multiple small exons that may alter the conformation of the extracellular region of the molecule. Differential use or switching of these small exons in conjunction with the differential expression of larger exons specifying regions associated with the cell membrane and cytoplasmic domains may signal key events in embryogenesis and histogenesis.     

Bryostatin analogue-induced apoptosis in mantle cell lymphoma cell lines

The anti-cancer effects of bryostatin-1, a potent diacylglycerol analogue, have traditionally been attributed to its action on protein kinase C. However, we previously documented apoptosis in a B non-Hodgkin lymphoma cell line involving diacylglycerol analogue stimulation of Ras guanyl-releasing protein, a Ras activator, and Bim, a proapoptotic Bcl-2 family protein. To further explore the role of Bim, we examined several Bim-deficient B non-Hodgkin lymphoma cells for their responses to pico, a synthetic bryostatin-1-like compound. The Bim(-) mantle cell lymphoma cell lines Jeko-1, Mino, Sp53, UPN1, and Z138 and the Bim(+) cell line Rec-1, as well as the Burkitt lymphoma cells lines BL2 (Bim(-)) and Daudi (Bim(+)), were examined for their response to pico using assays for proliferation and apoptosis as well as biochemical methods for Ras guanyl-releasing proteins and Bcl-2 family members. With the exception of UPN1, mantle cell lymphoma cell lines underwent pico-induced apoptosis, as did BL2. In some cases, hallmarks of apoptosis were substantially diminished in the presence of mitogen-activated protein kinase kinase inhibitors. Pico treatment generally led to increased expression of proapoptotic Bik, although the absolute levels of Bik varied considerably between cell lines. A pico-resistant variant of Z138 exhibited decreased Bik induction compared to parental Z138 cells. Pico also generally decreased expression of anti-apoptotic Bcl-XL and Mcl1. Although, these changes in Bcl-2 family members seem unlikely to fully account for the differential behavior of the cell lines, our demonstration of a potent apoptotic process in most cell lines derived from mantle cell lymphoma encourages a re-examination of diacylglycerol analogues in the treatment of this subset of B non-Hodgkin lymphoma cases.     

Modular structural units, exons, and function in chicken lysozyme.

By the application of the same algorithm for finding compact structural units encoded by exons as applied previously to hemoglobin, five units, M1-M5, were identified in chicken egg white lysozyme. They consist of residues 1-30, 31-55, 56-84, 85-108, and 109-129, respectively. I call these compact structural units "modules." As in hemoglobin, modules thus identified correspond well to exons--i.e., modules M1, M2 plus M3, M4, and M5 correspond to exons 1, 2, 3, and 4 of the lysozyme gene, respectively. Localization of the catalytic sites glutamic acid-35 and aspartic acid-52 on the module M2 suggests that this module might have worked as a functional unit in a primitive lysozyme. The good correspondence between exons and modules reinforces the idea of "proteins in pieces," which was derived from the fact of "genes in pieces." The evolutionary origin of the introns in globins and lysozyme is discussed.     

Identification of T cell lymphoma tumor antigens on human T cell lines.

All human T lymphoblast cell lines have been derived from subjects with leukemia secondary to thymic lymphoblastic lymphoma, a T cell malignancy, suggesting that such lines represent established cultures of neoplastic T cells. Based on this observation, we prepared rabbit antisera to T cell line HSB-2, removed reactivity for histocompatibility antigens and normal T cells by absorption with autocthonous B cell line CCRF-SB and normal thymocytes, and tested the absorbed antisera by complement-dependent cytotoxicity against a panel of normal and malignant cells. A representative antiserum reacted with all 4 T cell lines (mean cytotoxic index =56) and with tumor cells from 4 patients with T cell lymphoma (mean cytotoxic index = 50) but did not react with tumor cells from 6 patients with other types of leukemias (mean cytotoxic index = 2), with 3 B cell lines (mean cytotoxic index = 1), normal peripheral blood lymphocytes (mean cytotoxic index = 5), or normal thymocytes (mean cytotoxic index = 6). We conclude that appropriately absorbed antisera to human T cell lines detect T cell lymphoma tumor antigens.     

Anti-proliferative effect of interferon-gamma is enhanced by iron chelation in colon cancer cell lines in vitro

BACKGROUND/AIMS: The receptor of interferon-gamma (IFN-gammaR) consists of IFN-gammaR1 and R2. Resistance to the anti-proliferative effect of IFN-gamma is due to downregulation of IFN-gammaR2. The aim of this study was to investigate whether iron chelation could upregulate IFN-gammaR2 and enhance the anti-proliferative effect of IFN-gamma in colon cancer cell lines. METHODOLOGY: The colon cancer cell lines, SW480, COLO, and WiDr were treated with the iron chelating agent DFO, and the expression of IFN-gammaR1 and IFN-gammaR2 was evaluated by FACS. The anti-proliferative effect of IFN-gamma was investigated by MTT assay, and the proapoptotic effect was investigated by FACS with Annexin-V. RESULTS: FACS demonstrated that DFO increased the expression of IFN-gammaR2, whereas the effect on IFN-gammaR1 expression was less marked. MTT assay showed that cell growth was inhibited by DFO. Addition of DFO and IFN-gamma inhibited further, but inhibition was not observed with IFN-gamma alone. Apoptotic cells were increased by DFO, and further increased with DFO + IFN-gamma together. CONCLUSIONS: Expression of IFN-gammaR2 is restored by iron chelation, and the increased expression of IFN-gammaR2 enhances the anti-proliferative effect of IFN-gamma through induction of apoptosis in colon cancer cells.     

c-myc hypermutation is ongoing in endemic, but not all Burkitt's lymphoma

Deregulation of c-myc oncogene secondary to chromosomal translocation appears to play an essential role in the genesis of both endemic (African) Burkitt's lymphoma (eBL) and sporadic Burkitt's lymphoma (sBL). In most eBL, mutations in or near exon 1 disrupt normal c-myc regulatory sites. We examined c-myc sequences from a patient with sBL and two patients with eBL to determine (1) whether mutation is ongoing as the tumor clone expands, (2) the nature of mutations in the protein-coding exons 2 and 3, and (3) the extent of c-myc hypermutation in the two clinical forms of BL. Using the polymerase chain reaction (PCR), we amplified segments of c-myc from bulk tumor samples, cloned the products into plasmid vectors, and sequenced multiple subclones of each segment. The mutation frequencies in the control (remission bone marrow) and sBL tumor subclones were 0.65 x 10(-4) and 3.0 x 10(-4) (mutations/base), respectively (P greater than .25). Subclones from the two eBLs exhibited mutation frequencies of 20 x 10(-4) and 16 x 10(-4), respectively (P less than .001 v control). In addition to the consensus mutations seen in one eBL, a random pattern of unshared mutations was observed throughout c-myc in both samples, demonstrating that mutations may be introduced in a stepwise fashion. We noted a clear excess of transitions over transversions (30:9), which is qualitatively similar to the pattern observed in diverse examples of eukaryotic gene mutation. These data demonstrate that c-myc hypermutation is an ongoing process as the eBL tumor clone expands, is qualitatively different from immunoglobulin gene hypermutation, and is not a universal feature of BL, perhaps reflecting the nature of the translocation or the stage of tumor cell maturation.     

Human c-myc onc gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells.

Human sequences related to the transforming gene (v-myc) of avian myelocytomatosis virus (MC29) are represented by at least one gene and several related sequences that may represent pseudogenes. By using a DNA probe that is specific for the complete gene (c-myc), different somatic cell hybrids possessing varying numbers of human chromosomes were analyzed by the Southern blotting technique. The results indicate that the human c-myc gene is located on chromosome 8. The analysis of hybrids between rodent cells and human Burkitt lymphoma cells, which carry a reciprocal translocation between chromosomes 8 and 14, allowed the mapping of the human c-myc gene on region (q24 leads to qter) of chromosome 8. This chromosomal region is translocated to either human chromosome 2, 14, or 22 in Burkitt lymphoma cells.     

Constitutive production of colony-stimulating factor by mouse lymphoma cell lines is correlated with granulocytosis in vivo

The mouse lymphoma cell line Eb, its highly metastasizing variant ESb, and an unrelated metastasizing tumor MDAY-D2, were shown to produce colony-stimulating factor (CSF) constitutively both in vitro and in vivo in ascites. For each tumor, the amounts of CSF produced on a per-cell basis in vitro and in vivo were similar. The findings were substantiated using two different methods for CSF determination, a colony assay and an isotope incorporation test. Elevated levels of CSF in serum of mice with tumors were also found. Examination of blood from tumor-bearing mice revealed that whereas total leukocyte counts remained within the normal range, all three tumors caused a reversal of the normal neutrophil-lymphocyte ratio. The severity of the reversal correlated with the propensity of the tumor to elevate serum CSF rather than with in vitro CSF-producing capacity. Thus, whereas production of CSF in vivo was not related to the ability of the tumors to metastasize, it could be causative in creating an imbalance in normal hematopoiesis.     

Platelet receptor expression on three human megakaryoblast-like cell lines.

Platelets, the progeny of bone marrow megakaryocytes, are nonnucleated cells; many platelet proteins, including platelet membrane receptors, are believed to be derived from megakaryocytes. Several hematopoietic cell lines that exhibit megakaryocytic characteristics have been established as models for the study of megakaryocyte biology. We report here the screening of platelet receptor expression, in terms of functional coupling with the formation of two second messengers, calcium and cAMP, in three cell lines exhibiting megakaryoblastic properties: HEL, MEG-01, and DAMI. We show that all these cell lines respond to thrombin, ADP, epinephrine, and prostaglandin E1 (PGE1). However, transmembrane signaling pathways appear partly different from those present in mature platelets, because the action of thrombin was found to be positively coupled with the cAMP pathway, in addition to that of calcium, and because PGE1, which interacts with the cAMP pathway, also raises intracellular calcium levels in the three cell lines studied. Furthermore, an endothelin-1-induced increase in intracellular calcium level was observed in MEG-01 cells, strongly suggesting the expression of endothelin receptors on platelet precursors cells, whereas the presence of such receptors is controversial on platelets. These cell lines should prove useful in further studies of the expression and molecular pharmacology of platelet receptors on platelet precursor cells, as well as for the investigation of functional roles for platelet receptors on megakaryoblastic cells.     

Human papillomavirus type-related DNA and c-myc oncogene alterations in colon cancer cell lines

Although squamous-cell epithelium is the most frequent target site of human papillomavirus (HPV) infection, a similar infection is demonstrated in columnar epithelial cells in this paper. The papillomavirus expression in three cell lines was detected in colorectal adenocarcinoma of Chinese patients. The HPV-16 and HPV-18 DNA sequences were found in colorectal cancer cell lines, which might suggest the correlation of HPV to the etiology of colorectal cancers. In addition, c-myc oncogene was identified by amplification in all three colorectal cancer cell lines, but only normal germ-line fragments were found in control tissue. The correlation between HPV and c-myc, and the implications of these findings in colorectal cancers are also discussed.     

Possible DNA-RNA tumor virus interaction in human lymphomas: expression of retroviral proteins in Ramos lymphoma lines is enhanced after conversion with Epstein-Barr virus.

Epstein-Barr virus (EBV)-genome-negative human lymphoma lines, Ramos and BJAB, can be converted by EBV in vitro into EBV-genome-positive virus nonproducer lines. These cell lines have been used to identify surface antigens unique to EBV, with the expectation that such determinants might be related to the antigenic target responsible for EBV-specific immunosurveillance. Antisera prepared in rabbits immunized with either whole cells or purified plasma membranes were used in immunoblot experiments to analyze antigenic differences resulting from expression of the resident EBV genome. Unexpectedly, an increase in polypeptides of 32 and 70 kilodaltons was consistently observed in the EBV-converted Ramos lines. In contrast, these antigens were not expressed in BJAB or in its EBV-converted lines. These data suggested that p32 and gp70 might be murine leukemia virus (MuLV)-coded antigens because Ramos, but not BJAB, had been passaged in athymic nude mice during establishment of this cell line. This conclusion was confirmed by using antisera specific for MuLV p30 and gp70. Retroviral antigens were expressed constitutively at low levels in Ramos. Quantitative immunoblotting showed that EBV conversion of Ramos amplified the expression of MuLV proteins 3- to 5-fold. The molecular mechanism responsible for the enhanced expression is unknown. The biological relevance of this phenomenon is also not clear, but the interaction between a DNA and a RNA tumor virus in a Burkitt lymphoma line that carries both viruses may have important biological consequences in relation to retrovirus latency and tumor induction. These results also show that caution must be used when ascribing "uniqueness" to EBV-determined antigens, particularly in the Ramos lines. This warning extends also to the use of Ramos cell lines as immunogens, because immunization of rabbits elicited antibodies that recognized proteins of the same size as the retroviral antigens.     

Defects of FA/BRCA pathway in lymphoma cell lines

The aim was to find the possible relationship between defects in the FA/BRCA pathway of genomic maintenance and potential pathogenesis of T and B cell lymphoma. We screened 29 cell lines derived from diverse subtypes of lymphoma for possible FA pathway defects. The results indicated: no defect in FANCD2 ubiquitination, BRCA2 and FANCJ expression; absence of FANCN protein in three cell lines: HT, Sudhl4 and JEKO-1. This absence was correlated with enhanced MMC-induced G2 arrest, growth inhibition and high chromosomal breakage rate in the three cell lines. We only found one substitution in HT and JEKO-1 exon-5a fragment: c.1769C > T, p. A590V. But in another lymphoma cell line Sudhl4 with FANCN absence, we have not found any mutation. In conclusion, this mutation maybe the reason which caused FANCN protein expression absent or made the protein very unstable and lose its function in HT and JEKO-1 cell lines.     

Cellular foundation of curcumin-induced apoptosis in follicular lymphoma cell lines

OBJECTIVE: Although responsive to first-line treatments, follicular lymphoma (FL) remains a fatal disease of increasing worldwide incidence. In efforts to find novel approaches to inhibit proliferation and induce apoptosis in FL cells, we examined the action of naturally occurring compound curcumin in the three recently established FL cell lines. MATERIALS AND METHODS: Cytotoxic effects and determination of apoptotic attributes upon curcumin treatment were analyzed using growth inhibition, [(3)H]-thymidine, fluorescence microscopy, and flow cytometry assays, as well as Western blotting. Chemical inhibitor studies for the assessment of caspase and cathepsin contribution were applied. Expression of 10 members of the bcl-2 family proteins was evaluated by immunoblotting. RESULTS: Curcumin inhibited proliferation and growth, and induced profound apoptosis associated with a shift in the balance of the bcl-2 family proteins, in all cell lines tested. Strikingly, we observed that curcumin-induced caspase-dependent apoptosis is also associated with lysosomal rupture (LMP). An increase in intracellular ROS generation appeared critical for curcumin-evoked LMP, loss of Deltapsi(m,) caspase activation, and cell death, as well as ascorbic acid-mediated enhancement of curcumin's action. CONCLUSION: We have demonstrated for the first time that curcumin is an efficient inducer of apoptosis in FL cell lines, meriting its further evaluation in vivo.     

Exon cloning: immunoenzymatic identification of exons of the chicken lysozyme gene.

A 10-kilobase DNA fragment containing exons 1 and 2 of the chicken lysozyme gene has been randomly cleaved with DNase I. After tailing and cloning into the plasmid pUK230, Lac+ colonies were selected. Colonies harboring expressed fragments of the exons could be detected by an immunoenzymatic assay using antibodies against lysozyme. The smallest fragment coded for 10 amino acids and the largest coded for almost all residues of exon 2. These results suggest that any gene of any genome cloned in this way can be detected if antibodies against the gene product are available.