Coordinate suppression of myeloma-specific genes and expression of fibroblast-specific genes in myeloma X fibroblast somatic cell hybrids.

In most instances, fusion of differentiated cell types with fibroblasts has resulted in the extinction of the differentiation-specific traits of the non-fibroblast parental cell. To explore the genetic basis of this phenomenon, we have studied a series of somatic cell hybrids between mouse myeloma and fibroblasts. All the hybrids were adherent having a fibroblast-like phenotype. Molecular analysis revealed that plasma cell specific genes like the productively rearranged Ig genes, the J chain gene and genes for the cell surface markers CD20 and PC1, were extinguished in the hybrids. In contrast, fibroblast specific genes like fibronectin, alpha 2(I) and III collagens, as well as the receptor for fibroblast growth factor (flg), were expressed. Extinction was not due to chromosomal loss or lack of the relevant genes. To learn about the mechanism(s) of this phenomenon we have looked for the presence of positive and negative transcription factors in our hybrids. Expression of the PU.1 transcription factor, a member of the Ets transcription factor family normally expressed in B cells and macrophages, was lost in the cell hybrids. Interestingly, we found that the B-cell-specific Oct-2 transcription factor was still expressed at somewhat variable levels in several of the hybrid cell lines. In contrast, expression of the recently identified octamer coactivator BOB.1/OBF.1 was extinguished in all cell hybrids. This supports a critical role of this transcriptional coactivator for B-cell-specific gene expression. In addition, the Id and HLH462 genes coding for proteins known to repress bHLH transcription factors by formation of heterodimers, were found to be expressed at increased levels in fibroblasts and in the hybrids, indicating that their increased levels might also contribute to the suppression of myeloma-specific genes. Our results show that in myeloma x fibroblast hybrids, the phenotype of the fibroblast is dominant. It is suggested that fibroblasts contain regulatory "master" genes that are responsible for activation of the fibroblast differentiation pathway and suppress differentiation programs of other cell types.

A dynamic balance between ARP-1/COUP-TFII, EAR-3/COUP-TFI, and retinoic acid receptor:retinoid X receptor heterodimers regulates Oct-3/4 expression in embryonal carcinoma cells.

The Oct-3/4 transcription factor is a member of the POU family of transcription factors and, as such, probably plays a crucial role in mammalian embryogenesis and differentiation. It is expressed in the earliest stages of embryogenesis and repressed in subsequent stages. Similarly, Oct-3/4 is expressed in embryonal carcinoma (EC) cells and is repressed in retinoic acid (RA)-differentiated EC cells. Previously we have shown that the Oct-3/4 promoter harbors an RA-responsive element, RAREoct, which functions in EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for positive regulators of transcription and in RA-differentiated EC cells as a binding site for negative regulators. Our present results demonstrate that in P19 and RA-treated P19 cells, the orphan receptors ARP-1/COUP-TFII and EAR-3/COUP-TFI repress Oct-3/4 promoter activity through the RAREoct site in a dose-dependent manner. While the N-terminal region of the ARP-1/COUP-TFII receptor is dispensable for this repression, the C-terminal domain harbors the silencing region. Interestingly, three different RA receptor:retinoid X receptor (RAR:RXR) heterodimers, RAR alpha:RXR alpha, RAR beta:RXR alpha, and RAR beta:RXR beta, specifically bind and activate Oct-3/4 promoter through the RAREoct site in a ligand-dependent manner. We have shown that antagonism between ARP-1/COUP-TFII or EAR-3/COUP-TFI and the RAR:RXR heterodimers and their intracellular balance modulate Oct-3/4 expression. Oct-3/4 transcriptional repression by the orphan receptors can be overcome by increasing amounts of RAR:RXR heterodimers. Conversely, activation of Oct-3/4 promoter by RAR:RXR heterodimers was completely abolished by EAR-3/COUP-TFI and by ARP-1/COUP-TFII. The orphan receptors bind the RAREoct site with a much higher affinity than the RAR:RXR heterodimers. This high binding affinity provides ARP-1/COUP-TFII and EAR-3/COUP-TFI with the ability to compete with and even displace RAR:RXR from the RAREoct site and subsequently to actively silence the Oct-3/4 promoter. We have shown that RA treatment of EC cells results in up-regulation of ARP-1/COUP-TFII and EAR-3/COUP-TFI expression. Most interestingly, in RA-treated EC cells, the kinetics of Oct-3/4 repression inversely correlates with the kinetics of ARP-1/COUP-TFII and EAR-3/COUP-TFI activation. These findings are in accordance with the suggestion that these orphan receptors participate in controlling a network of transcription factors, among which Oct-3/4 is included, which may establish the pattern of normal gene expression during development.

Retinoic acid represses Oct-3/4 gene expression through several retinoic acid-responsive elements located in the promoter-enhancer region.

The Oct-3/4 gene product, which belongs to the POU family of transcription factors, is a good candidate for regulating initial differentiation decisions. It is expressed in the earliest stages of embryogenesis and repressed in subsequent stages. Retinoic acid (RA)-induced differentiation of embryonal carcinoma (EC) cells is accompanied by decreased expression of the Oct-3/4 gene. Previous findings show that sequences in the Oct-3/4 enhancer region (designated RARE1) are targets for RA-mediated repression (H. Okazawa, K. Okamoto, F. Ishino, T. Ishino-Kaneko, S. Takeda, Y. Toyoda, M. Muramatsu, and H. Hamada, EMBO J. 10:2997-3005, 1991). Our present results demonstrate conclusively that the TATA-less Oct-3/4 promoter is also a target for RA-induced repression. We identified a novel cis element in the Oct-3/4 promoter harbors a putative Sp1 binding site and a RA-responsive element (designated RAREoct), which are juxtaposed to one another. Protein binding to the Sp1 site is independent of protein binding to the RAREoct sequence. Unlike the RARE1 situated in the Oct-3/4 enhancer which does not contain a typical RAR recognition site, the RAREoct identified in this study consists of three directly repeated motifs that exhibit extensive homology to RARE sequences located in RA-responsive genes. Moreover, the RAREoct shows different DNA-binding characteristics and DNase I footprint patterns with nuclear proteins isolated from undifferentiated versus RA-differentiated EC cells. This suggests that the RAREoct element binds different nuclear proteins in RA-treated and untreated EC cells which most probably belong to the RA receptor, retinoid X receptor, or orphan receptor families of transcription factors. Using site-directed mutagenesis, we show that the RAREoct contributes to the transcriptional activation of Oct-3/4 promoter in P19 cells and, most interestingly, mediates the RA-induced repression in RA-differentiated EC cells. Thus, the RAREoct element could be one of the points of integration of several signalling pathways influencing Oct-3/4 expression. In accordance with the suggestion that suppression of Oct-3/4 expression is a crucial step during embryogenesis, the Oct-3/4 upstream region contains multiple targets for RA-induced repression, probably to ensure accurate and prompt repression of Oct-3/4 expression. It is possible that these repressors are differentially used at specific stages of development in response to various signals.

Production of heparanase by normal and neoplastic murine B-lymphocytes.

The production of heparanase, an endoglycosidase capable of degrading heparan sulfate from the subendothelial extracellular matrix (ECM), was investigated in various murine B-lymphoid tumors representing distinct maturation stages of the B-cell lineage. We found that heparanase is produced and released by 3 out of 4 pre-B lymphomas and by 4 B lymphomas examined. In contrast, 5 plasmacytomas and resting normal B lymphocytes, expressed little, if any, heparanase activity. Treatment with LPS resulted in high expression of the enzyme by normal B-lymphocytes, but there was no effect on the constitutive production of heparanase by myeloma or B-lymphoma cells. Our results indicate that heparanase is produced by B cells during discrete stages of their maturation. We suggest that heparanase may play a role in B-cell migration by enabling pre-B and B lymphocytes to leave the bone-marrow compartment and recirculate among peripheral lymphoid organs.

Extinction of Ig genes expression in myeloma x fibroblast somatic cell hybrids is accompanied by repression of the oct-2 gene encoding a B-cell specific transcription factor.

In most instances, fusion of differentiated cell types with fibroblasts has resulted in the extinction of differentiation-specific traits of the nonfibroblast parental cell. To explore the genetic basis of this phenomenon, we have used a series of somatic cell hybrids between myeloma cells and fibroblasts. Previous findings show that in these hybrids expression of the immunoglobulin (Ig) genes was extinguished at the transcriptional level. Our present results show that NF-kappa B transcription factor, known to be critical for kappa-chain enhancer activity, is present although in a lower amount, in the nucleus and in the cytosolic fraction of most of these hybrids (probably attached to the previously postulated I-kappa B inhibitor). In contrast, the expression of the NF-A2/OTF-2 transcription factor encoded by the oct-2 gene, which binds to the octameric motif located in the Ig promoters and heavy chain gene enhancer, is extinguished at the transcriptional level. Our data thus suggest that extinction of Ig genes expression occurs via an indirect mechanism in which a fibroblast factor suppresses transcription factor(s) which are critical for Ig transcription.

Mother- and father-preterm infant relationship in the hospital preterm nursery.

The relationships of Israeli mothers and fathers with their 38 preterm infants during hospitalization were traced in a short-term longitudinal study. Parent-infant interactions were observed and self-reports of parental feelings and perceptions were assessed twice: at the beginning and end of the nursery period. Mothers engaged in more caregiving, talking, and holding during initial contacts, but the disparity in maternal and paternal interactions decreased with time. Except for caregiving, in which mothers still surpassed fathers, fathers equaled mothers in all other activities at the time of the infants' discharge from the hospital. Fathers consistently surpassed mothers in playing and stimulating. Mothers perceived their infants to be more difficult than did fathers but reported enjoying them more. With time, parents were less disappointed and concerned over the infants' well-being but perceived them as more difficult. The data also demonstrated an association between infant behavioral states, parental feelings and perceptions, and parental behavior.

Extinction of expression of the translocated myc gene in somatic cell hybrids between mouse myeloma and L-cells.

Most murine plasma-cell tumors show a t(12;15) reciprocal chromosomal translocation which truncates the first exon of one of the myc gene alleles and fuses it to one of the switch regions of the immunoglobulin (Ig) heavy-chain locus. This results in constitutive activation of the translocated myc gene and the production of smaller-sized mRNA molecules, which are initiated at new sites in the first myc intron. The normal myc allele is not expressed in these myeloma cells. We have studied the expression of the translocated myc gene in somatic cell hybrids between mouse myeloma and L-cells. Our previous findings show that Ig gene expression is extinguished in such hybrids. In the present work we found that the hybrids contain the normal and translocated myc genes. In contrast to the myeloma parental cells which express the translocated myc gene, the hybrids are similar to the L-cells in expressing only the normal myc allele. Our results suggest that the L-cell, fibroblast-like phenotype, is dominant in these hybrids, and show that the translocated myc gene is expressed in a tissue-specific manner in the context of the myeloma cell, and is not expressed when subjected to a fibroblast-like cellular environment.