Quantitative analysis of chromosomal CGH in human breast tumors associates copy number abnormalities with p53 status and patient survival.

We present a general method for rigorously identifying correlations between variations in large-scale molecular profiles and outcomes and apply it to chromosomal comparative genomic hybridization data from a set of 52 breast tumors. We identify two loci where copy number abnormalities are correlated with poor survival outcome (gain at 8q24 and loss at 9q13). We also identify a relationship between abnormalities at two loci and the mutational status of p53. Gain at 8q24 and loss at 5q15-5q21 are linked with mutant p53. The 9q and 5q losses suggest the possibility of gene products involved in breast cancer progression. The analytical techniques are general and also are applicable to the analysis of array-based expression data.

Regulation of growth in a neoplastic B cell line by transfected subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinase.

cAMP inhibits the proliferation of both normal peripheral blood B and T lymphocytes as well as the proliferation of a human neoplastic B precursor cell line (Reh). To positively show that this is mediated via the the catalytic subunit, C alpha, of cAMP-dependent protein kinase, stably transfected Reh cell lines overexpressing C alpha were established. This was achieved by transfection with a construct confering hygromycin resistance together with a zinc-inducible expression of C alpha from the human metallothionine promoter. C alpha transfected clones were shown to confer a 2- to 2.5-fold zinc-dependent increase in C alpha messenger RNA, immunoreactive C, and phosphotransferase activity. The growth rate of clones transfected with C alpha was retarded, and a zinc-dependent inhibition of cell proliferation was demonstrated in the presence of a small trigger dose of forskolin. In contrast, overexpression of the regulatory subunit I alpha had no effect on cAMP-dependent inhibition of cell proliferation. Furthermore, expression of mutant regulatory subunit I alpha AB, which renders cAMP-dependent protein kinase unresponsive to cAMP, clearly protected against that inhibitory effect of cAMP. These data provides evidence that activation of the C subunit (C alpha) of cAMP-dependent protein kinase mediates the inhibitory action of cAMP on cell proliferation in Reh cells.

CDw75 antigen expression in breast lesions.

Alterations on the cell surface of the oligosaccharide portion of glycoproteins and glycolipids are thought to play a role in tumorigenesis. Sialyltransferase catalyzes the incorporation of sialic acid to the carbohydrate group of glycoconjugates. Sialyltransferase has been found elevated in different tumour tissues and in the serum of cancer patients. In the present study we have examined the expression of the beta-galactosyl alpha 2,6-sialyltransferase requiring epitope CDw75, with the monoclonal antibody HH2. 142 breast lesions were included. 21% of the carcinomas in situ and 35% of the invasive carcinomas showed a diffuse cytoplasmic staining. Seven cases of invasive carcinomas also showed a distinct membrane immunoreactivity. We found no correlation between reactivity for CDw75 in malignant lesions and their metastatic potential. Only five out of 11 primary tumours with metastases expressed CDw75 in the primary tumour. In the benign lesions, there was a positive reaction in proliferating lesions, e.g. intraductal papillomas (2 out of 3 cases) and in epithelial proliferations in fibrocystic disease (10 out of 14 cases). None of the four fibroadenomas and phyllodes tumours and only one out of 22 cases of normal breast tissues showed immunoreactivity for HH2. In the malignant lesions, CDw75 was more frequently expressed in the carcinomas of high malignancy grade. The high frequency of immunoreactivity among the benign breast lesions can be indicative of activation of the epithelial cells.

Expression of CD18 (integrin beta 2 chain) correlates with prognosis in malignant B cell lymphomas.

Several studies have shown that cell cycle related parameters including DNA synthesis and activation antigen expression can predict patient survival in lymphoma patients. In this study of 69 malignant B cell lymphomas we have examined the cell surface expression of several cell interaction and activation molecules by flow cytometry. Expression of CD18 (integrin beta 2 chain) was found to correlate strongly with patient survival (median follow up 50 months) even when adjusting for other important prognostic factors (P = 0.0001). The percentage of cells positive for CDw75 proved important both as a single parameter and in the multivariate analysis. Histology, classified as low versus high grade malignancy, bulky versus not bulky disease and high versus low thymidine incorporation, was also found to correlate with prognosis in this study.

The retinoblastoma gene product is bound in the nucleus in early G1 phase.

The product of the retinoblastoma susceptibility gene (pRB) exerts its growth-regulatory effects during the G1 phase of the cell cycle, where all pRB present has been assumed to be in the underphosphorylated form. We demonstrate here that pRB is underphosphorylated and firmly bound in the nucleus only in early G1 phase. All G0 cells contain bound, underphosphorylated pRB. The duration of the cell cycle and of the G1 phase seems to be determined by the time during which pRB is underphosphorylated and bound in the nucleus. The observed time lag between the phosphorylation and release of pRB in the G1 phase and entry into S phase was 6.5 h and independent of the G1 transit time. The data suggest that pRB is not directly involved in initiation of DNA replication.

Cell cycle-dependent regulation of CDw75 (beta-galactoside alpha-2,6-sialyltransferase) on human B lymphocytes.

Within the hematopoietic system, CDw75 is primarily expressed on cells of the B cell lineage. Cloning and sequencing of the gene has shown CDw75 to be a beta-galactoside alpha-2,6-sialyltransferase. This enzyme plays an important role in the intracellular terminal glycosylation pathways in various cell types. In this article, we demonstrate that COS cells transfected with the CDw75 cDNA clone displayed sialyltransferase activity, in contrast to mock-transfected cells. We also found that activated B cells displayed an increased enzyme activity compared to resting cells, in accordance with the staining data. Moreover, CDw75 expression was found to be up-regulated approximately 7-9-fold from early G1 to the G2/M phases of the cell cycle in peripheral blood leukocyte B cells. This was shown by staining of in vitro activated B cells with the anti-CDw75 monoclonal antibody HH2, using cell fractions corresponding to different stages of the cell cycle. Using a combination of Hoechst 33258 and propidium iodide after bromodeoxyuridine incorporation, it is possible to distinguish between different phases of the first and second cell cycle. By combining this with HH2 immunofluorescence staining, using a multistation multiparameter flow cytometry program, we confirmed the cell cycle-dependent expression of CDw75. Immunocytochemical stainings of cytospin specimens of elutriated B cells showed that the antigen was up-regulated in late G1 before the appearance of the nuclear activation antigen Ki67. Finally, we showed that activated B cells secreted soluble CDw75 into the medium, as demonstrated by a specific blocking of HH2 staining of B cells using suboptimal concentrations of HH2. In accordance with this, we observed small, but detectable levels of soluble sialyltransferase activity in supernatants of activated B cells.

A new method for detachment of Dynabeads from positively selected B lymphocytes.

This paper describes a method for the detachment of immunomagnetic beads from positively selected human B lymphocytes. After rosetting of B cells using anti-CD19 coated magnetic beads (Dynabeads M-450 Pan B, Dynal), the Dynabeads were rapidly detached (efficiency 80%) from the cells using goat anti-mouse-Fab antiserum (DETACHaBEAD, Dynal) at ambient temperature. Isolated B cells did not show significant differences in the expression of a number of B cell antigens when compared to B cells stained in fresh whole blood. In contrast, positively selected B cells that had detached from the beads following overnight incubation, demonstrated a significantly reduced expression of certain of the antigens examined (CD19, CD20 and CD23). It was further demonstrated that neither anti-CD19 nor anti-Fab resided on the surface of the cells after detachment. The cells were still in G0 phase (greater than 90%) at the end of the isolation procedure. Moreover, anti-IgM antibodies stimulated the vast majority of the cells to leave the G0 phase, and to progress through S phase in the presence of growth factors. The cells could also be stimulated to differentiate, further confirming the normal functional capacity of the isolated cells. The method described in this paper can also be used for the detachment of other positively selected cells, such as CD4+ T cells, CD8+ T cells and CD34+ stem cells.

Interleukin-7 differentiates a subgroup of acute lymphoblastic leukemias.

The bone marrow stromal cell-derived growth factor interleukin-7 (IL-7) is known to stimulate growth of normal human B-cell precursors. In the present report, we have examined the effect of IL-7 on neoplastic B-cell precursors. Leukemic cells from 20 patients with common acute lymphoblastic leukemia (ALL) were highly purified by removing contaminating T cells and monocytes by rosetting with immunomagnetic beads. IL-7 markedly reduced the DNA synthesis in leukemic cells from three patients. This inhibition of DNA synthesis was accompanied by maturation of the cells, as demonstrated by the induced expression of the differentiation antigens CD19, CD20, CDw75, and surface mu-chain, and a decreased expression of terminal deoxynucleotidyl transferase. By examining G1 parameters, such as MYC, 4F2, and transferrin-receptor levels analyzed by flow cytometry as well as RNA and the cell cycle regulated antigen Ki67, it appeared that the cells were inhibited late in G1. Leukemic cells from the majority of the cases (12 of the 20 patients) responded to IL-7 with enhanced DNA synthesis without detectable maturation, as has been reported for their normal counterparts. Low molecular weight B-cell growth factor greatly potentiated the IL-7-induced growth stimulation of these cells. Thus, we have shown that IL-7 is capable of inhibiting proliferation of leukemic cells isolated from a subgroup of ALLs, and that this growth inhibition is accompanied by maturation of the cells.

Independent regulation of 55-kDa and 75-kDa tumor necrosis factor receptors during activation of human peripheral blood B lymphocytes.

We have studied the expression of two different tumor necrosis factor receptors (TNFR; 55 kDa and 75 kDa) on resting and activated human peripheral blood B lymphocytes using specific monoclonal antibodies (mAb). Flow cytometric analysis revealed that most resting B cells expressed small amounts of the 75-kDa TNFR, and that the 75-kDa TNFR was markedly up-regulated upon stimulation with anti-mu or Staphylococcus aureus Cowan strain I (SAC). In contrast, the expression of the 55-kDa TNFR was low on resting as well as on activated cells. B cell activation was accompanied by an increased binding of biotinylated TNF-alpha, and this binding could be blocked by preincubation by utr-1 (anti-75-kDa TNRF), but not the htr (anti-55-kDa TNFR) antibodies. Notably, a number of cytokines tested (interleukin 1 to 8, interferon-gamma, TNF-alpha and -beta) did not influence the expression of either the 75-kDa or the 55-kDa TNFR when given to resting B cells. Moreover, phorbol 12-myristate 13-acetate led to an early, marked down-regulation of the 75-kDa TNFR expression, followed by a later modest increase after greater than 24 h. In contrast to other cell systems where htr mAb have been found either to mimic or to inhibit TNF action, htr mAb had insignificant effects in assays for restimulation of preactivated B cells. However, utr-1 markedly inhibited the TNF-beta but only partly inhibited the TNF-alpha-induced proliferation. Taken together, our data suggest that changes in 75-kDa protein expression is responsible for the increased TNFR expression on activated vs. resting peripheral blood B cells and that this protein also may play an important functional role.

Use of size fractionation of in vitro-activated human B lymphocytes for studies of cell cycle-dependent growth regulation.

Cell cycle progression of in vitro-stimulated human B lymphocytes occurs asynchronously. In order to allow detailed studies of growth control in G1, B cells were stimulated with anti-mu and low molecular weight B-cell growth factor (LMW BCGF) for 50 h and subsequently separated into nine fractions of cells by means of centrifugal elutriation. As judged by volume profiles, activation antigen expression and DNA content, the cells in fractions 1-4 were in early to mid-G1, while fractions 5-7 mainly contained cells in late G1, and fractions 8-9 contained cells mainly in S and G2. Cells in fractions 5-7 had passed the commitment point, as demonstrated by a high spontaneous incorporation of [3H]thymidine when recultured in medium alone. Moreover, S-phase entry of these cells was largely unaffected by exogenous growth-promoting or growth-inhibitory signals. Cells in early (fractions 1-2) and intermediate fractions (fractions 3-4) showed a negligible spontaneous [3H]thymidine incorporation, but a significant proportion of these cells progressed to S phase upon restimulation. Moreover, while IL-4 or the anti-CD40 MoAb G28-5 potently stimulated cells in early and intermediate fractions, the responsiveness of LMW BCGF alone was obtained just prior to the commitment point.