Expression of the Bcl-2 family member A1 is developmentally regulated in T cells.

During T cell development, cells that fail to meet stringent selection criteria undergo programmed cell death. Thymocyte and peripheral T cell susceptibility to apoptosis is influenced by expression of Bcl-2 family members, some of which are expressed in a developmentally patterned manner. We previously showed developmentally regulated expression of A1, an anti-apoptotic Bcl-2 family member, among B cell developmental subsets. Here we show that cells of the T lineage also express A1 in a developmentally regulated manner. Both A1 mRNA and A1 protein are readily detectable in the thymus, and while present among DN cells, A1 mRNA is up-regulated to very high levels among double-positive (DP) thymocytes. It is then down-regulated to moderate levels among single-positive (SP) thymocytes, and finally expressed at approximately 25-fold lower levels among mature SP CD4(+) and CD8(+) lymph node T cells than among DP thymocytes. Furthermore, we find that in vitro TCR ligation up-regulates A1 expression among both DP and SP thymocytes. Together, these data show that A1 expression is developmentally regulated in T lymphocytes and is responsive to TCR signaling, suggesting that A1 may play a role in maintaining the viability of DP thymocytes.

Long-lived B cells are distinguished by elevated expression of A1.

Only 5% of the 15 million B cells formed daily reach the long-lived peripheral B cell pool, presumably reflecting both negative and positive selection. These selective events occur primarily during late stages of differentiation in the marrow and periphery, when newly formed B cells bear surface IgM (sIgM), but differ from mature B cells in their expression of heat-stable Ag (CD24), B220 (CD45), and sIgD. Because genes of the Bcl-2 family influence longevity, we compared the expression of Bcl-2, Bax, and A1 among immature vs mature peripheral B cells using semiquantitative reverse-transcriptase PCR. While the levels of both Bcl-2 and Bax mRNA remain constant in these two populations, A1 expression is strikingly up-regulated among mature B cells. In addition, A1 expression is low among pro- and pre-B cells, as well as in immature (sIgM+) marrow B cells. Together, these data indicate that A1 mRNA expression is low at all stages of B cell development before final maturation in the periphery and, unlike other Bcl-2 family members whose expression changes little after marrow egress, A1 is up-regulated 10-fold as cells are recruited into the long-lived peripheral B cell pool.

Human neuroblastoma cell lines regain catecholamine fluorescence when xenografted into athymic (nude) mice.

Detection of catecholamine production by neuroblastoma is a useful tumor marker. The majority of neuroblastoma patients have elevated levels of urinary catecholamines and/or their metabolites, and have tumors, which show histochemical evidence of catecholamines using glyoxylic acid-induced catecholamine fluorescence. By contrast, continuous cell lines derived from neuroblastomas lack catecholamine fluorescence in vitro. In this study, we report that 11 out of 12 human neuroblastoma cell lines established from catecholamine-positive tumors displayed histochemical evidence of catecholamines when grown as xenografts in athymic (nude) mice. Catecholamine fluorescence in these xenograft tumors decayed over a 5 day period when the cells were placed into tissue culture. Xenograft tumors of cell lines derived from four catecholamine-negative neuroblastomas or seven primitive neuroectodermal tumors (PNET) did not show catecholamine fluorescence. Ultrastructural comparisons of cell lines in vitro with their corresponding tumors in vivo showed that six of eight cell lines had fewer dense core (neurosecretory) granules in vitro compared to the more readily detectable dense core granules seen in nude mouse tumor tissue. These data indicate that catecholamine synthesis and/or storage in human neuroblastoma cells requires factor(s) not present in the in vivo environment. As neuroblastoma cell lines derived from catecholamine-positive tumors retain the ability to produce and store catecholamines in vivo, such cell lines can be used to identify factors critical to catecholamine production in human neurons.

Human neuroblastoma cell lines that express N-myc without gene amplification.

BACKGROUND: About one half of aggressive neuroblastomas lack N-myc amplification. Cell lines from such tumors are needed to determine the biological basis of aggressive tumor behavior. METHODS: Neuroblastoma cell lines were established from a primary tumor (SMS-LHN) and a bone marrow metastasis (LA-N-6) of two children with Stage IV neuroblastoma. Although both cell lines and their original tumors lacked N-myc genomic amplification, these patients died of progressive disease. RESULTS: SMS-LHN and LA-N-6 can be distinguished from primitive neuroectodermal tumor (PNET) lines by cell surface antigen expression and catecholamine production. Cytogenetic analysis of each cell line revealed unique genetic rearrangements, whereas both lines showed abnormalities involving chromosome 2. Neither cell line contained double-minute chromosomes, homogeneously staining regions, a 1p chromosomal deletion, or t(11;22). The growth rates of these two new lines in vitro and in vivo (as xenografts in nude mice) are slower than N-myc amplified neuroblastoma lines. Both lines express greater amounts of N-myc RNA and protein relative to nonneuroblastoma cell lines (including PNET), although not to the extent of cell lines with N-myc genomic amplification. CONCLUSIONS: The relatively large amount of N-myc expression in these two new cell lines suggests that N-myc expression without amplification could play a role in the pathogenesis of some neuroblastomas. These cell lines should be useful for investigating mechanisms and consequences of N-myc gene activation other than genomic amplification.

Determination of subcutaneous tumor size in athymic (nude) mice.

The athymic (nude) mouse is a useful model for studying the biology and response to therapies of human tumors in vivo. A survey of recent literature revealed the use of 19 different formulas for determining the size of subcutaneous tumors grown as xenografts in nude mice (2 for determining tumor area, 3 for tumor diameter, and 14 for calculating tumor volume). We compared the volumes, areas, and diameters predicted by each of the 19 formulas with the actual weights of 50 tumors ranging from 0.46 to 22.0 g established in nude mice as xenografts from human cell lines. In addition to determining how well each formula predicted relative tumor size, we analyzed how well each formula estimated actual tumor mass. The ellipsoid volume formulas (pi/6 x L x W x H and 1/2 x L x W x H) were best for estimating tumor mass (r = 0.93), whereas measurements of diameter correlated poorly with tumor mass (r less than 0.66). Although determination of tumor area correlated well with tumor mass in small tumors (r = 0.89), correlations of area with tumor mass for large tumors were poor (r = 0.41). We conclude that determination of the ellipsoid volume from measurements of three axes consistently yields the most accurate estimations of both relative and actual tumor mass.