Kenneth B. Marcu

Posttranscriptional mechanisms are responsible for accumulation of truncated c-myc RNAs in murine plasma cell tumors

c-myc Messenger RNAs are known to be extremely unstable (t1/2 = 10 min) in normal and tumor cells, suggesting that degradation could play an important role in regulating their steady state level in the cytoplasm. We have investigated the stabilities of c-myc mRNAs in three murine plasmacytomas, where the c-myc gene either remains intact (ABPC20) or exists in a truncated form (MPC-11 and J558L) subsequent to 6;15 or 12;15 chromosome translocations respectively, and in an A-MuLV-induced pre-B lymphoma line (18-81.5) that lacks chromosome translocations and contains both c-myc genes in their normal context. The truncated myc genes in J558L and MPC-11 lack the promoters of the normal gene but are transcribed from cryptic promoters within the first c-myc intron. We found that posttranscriptional processes are largely responsible for the higher steady state accumulations of truncated c-myc transcripts, while broken and intact c-myc genes are transcribed at comparable rates.

Novel NEMO/IκB Kinase and NF-κB Target Genes at the Pre-B to Immature B Cell Transition

The IκB kinase (IKK) signaling complex is responsible for activating NF-κB-dependent gene expression programs. Even though NF-κB-responsive genes are known to orchestrate stress-like responses, critical gaps in our knowledge remain about the global effects of NF-κB activation on cellular physiology. DNA microarrays were used to compare gene expression programs in a model system of 70Z/3 murine pre-B cellsversus their IKK signaling-defective 1.3E2 variant with lipopolysaccharide (LPS), interleukin-1 (IL-1), or a combination of LPS + phorbol 12-myristate 13-acetate under brief (2 h) or long term (12 h) stimulation. 70Z/3-1.3E2 cells lack expression of NEMO/IKKγ/IKKAP-1/FIP-3, an essential positive effector of the IKK complex. Some stimulated hits were known NF-κB target genes, but remarkably, the vast majority of the up-modulated genes and an unexpected class of repressed genes were all novel targets of this signaling pathway, encoding transcription factors, receptors, extracellular ligands, and intracellular signaling factors. Thirteen stimulated (B-ATF, Pim-2, MyD118,Pea-15/MAT1, CD82, CD40L,Wnt10a, Notch 1, R-ras,Rgs-16, PAC-1, ISG15, andCD36) and five repressed (CCR2,VpreB, λ5, SLPI, andCMAP/Cystatin7) genes, respectively, were bona fide NF-κB targets by virtue of their response to a transdominant IκBαSR (super repressor). MyD118 andISG15, although directly induced by LPS stimulation, were unaffected by IL-1, revealing the existence of direct NF-κB target genes, which are not co-induced by the LPS and IL-1 Toll-like receptors.

5′ Flanking region of immunoglobulin heavy chain constant region genes displays length heterogeneity in germlines of inbred mouse strains

Genomic Southern blotting analysis reveals that a 5 flanking DNA sequence located 1.5-2.0 kb upstream from the C mu and C alpha genes undergoes extensive length variation in the germlines of inbred and wild mice. In addition, genomic clones of the BALB/c C mu gene undergo deletion events during their propagation in Charon 4A when maintained in RecA+ bacterial strains. These deletion events occur between the JH cluster and the C mu gene and approximately 2.0 kb 5 of the C alpha gene, and may encompass several kb of DNA. Deletion events are capable of yielding a group of C mu clones which differ in size by units of 100-200 bp. The site of length variation in the mouse germline corresponds to the location of these cloning-derived deletion events, suggesting that both phenomena are reflections of intrinsic genetic properties of these Ig gene 5 flanking sequences.

Recombinant IκB Kinases α and β Are Direct Kinases of IκBα

Activation of the transcription factor NF-κB is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IκB. A large multiprotein complex, the IκB kinase (IKK), catalyzes the phosphorylation of IκB. The two kinase components of the IKK complex, IKKα and IKKβ, were overexpressed in insect cells and purified to homogeneity. Both purified IKKα and IKKβ specifically catalyzed the phosphorylation of the regulatory serine residues of IκBα. Hence, IKKα and IKKβ were functional catalytic subunits of the IKK complex. Purified IKKα and IKKβ also preferentially phosphorylated serine as opposed to threonine residues of IκBα, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKKα and IKKβ revealed that the kinase activity of IKKβ on IκBα is 50–60-fold higher than that of IKKα. The primary difference between the two activities is the K m for IκBα. The kinetics of both IKKα and IKKβ followed a sequential Bi Bi mechanism. No synergistic effects on IκBα phosphorylation were detected between IKKα and IKKβ. Thus, in vitro, IKKα and IKKβ are two independent kinases of IκBα.

Synergism of v-myc and v-Ha-ras in the in vitro neoplastic progression of murine lymphoid cells.

Murine bone marrow was either singly or doubly infected with retroviral vectors expressing v-myc (OK10) or v-Ha-ras. The infected bone marrow was cultured in a system that supports the long-term growth of B-lineage lymphoid cells. While the v-myc vector by itself had no apparent effect on lymphoid culture establishment and growth, infection with the v-Ha-ras vector or coinfection with both v-myc and v-Ha-ras vectors led to the appearance of growth-stimulated cell populations. Clonal pre-B-cell lines stably expressing v-Ha-ras alone or both v-myc and v-Ha-ras grew out of these cultures. In comparison with cell lines expressing v-Ha-ras alone, cell lines expressing both v-myc and v-Ha-ras grew to higher densities, had reduced dependence on a feeder layer for growth, and had a marked increase in ability to grow in soft-agar medium. The cell lines expressing both oncogenes were highly tumorigenic in syngeneic animals. These experiments show that the v-myc oncogene in synergy with v-Ha-ras can play a direct role in the in vitro transformation of murine B lymphoid cells.

Mouse rDNA nontranscribed spacer sequences are found flanking immunoglobulin CH genes and elsewhere throughout the genome

When a cloned 6 kb Eco RI-Sal I fragement of mouse ribosomal gene nontranscribed spacer DNA (rDNA NTS) was used to screen a BALB/c mouse gene library, 25% of the recombinant phage hybridized with it. In situ hybridization experiments and characterization of 12 clones selected using this probe supported the idea that sequences homologous to this rDNA NTS region are scattered throughout the genome. Subsequently, sequences homologous to mouse rDNA NTS were found flanking mouse mu, alpha and gamma 2b immunoglobulin CH genes. One region was localized 3 to the mu coding sequence, an area which has been identified as an intervening sequence between the secreted C mu heavy chain terminus and the C terminal portion of the membrane-bound C mu heavy chain.

c-myc and Functionally Related Oncogenes Induce Both High Rates of Sister Chromatid Exchange and Abnormal Karyotypes in Rat Fibroblasts

Activated myc genes are found in a broad variety of spontaneous malignancies in rodents and humans as well: Burkitt Lymphoma (Klein and Klein 1985; Croce et al. 1984; Dalla-Favera et al. 1985; this volume), neuroblastoma (Schwab 1985), small cell lung carcinoma (Nau et al. 1985), mouse plasmacytoma (Klein and Klein 1985; this volume) and rat immunocytoma (Pear, this volume).

Synergy of an IgH Promoter-Enhancer-Driven c-myc/v-Ha-ras Retrovirus and Pristane in the Induction of Murine Plasmacytomas

Intraperitoneal injection of BALBc/An mice with a single 0.5 ml dose of the mineral oil pristane leads to the appearance of malignant Ig-secreting (usually IgA) plasma cells in 25% of mice after minimal latent periods of 120 days and mean latent periods between 210–220 days (Potter and Wax 1983). Over 95% of these plasmacytomas (PCTs) contain rcpt(12;15) or rcpt(6;15) chromosomal translocations in which the c-myc gene on chromosome 15 is juxtaposed with immunoglobulin heavy or light chain loci on chromosomes 12 and 6 respectively. These structural alterations disrupt the normal regulation of the c-myc gene which is thought to be causally related to the development of malignancy (reviewed in Potter 1984). Indeed infection of pristane primed mice with wild-type Moloney (Mo-MuLV) helper virus and the replication defective J-3 retrovirus, which contains a partially deleted raf oncogene and a hybrid MH2/MC29 avian v-myc oncogene induced shortened latency plasmacytomas which lacked c-myc translocations (Potter et. al. 1987). On the other hand experiments which used murine retroviruses harboring an LTR driven murine c-myc gene only induced myeloid malignancies in pristane primed mice (Wolff et. al. 1986).

An In Vitro Model for Tumor Progression in Murine Lymphoid Cells

Histopathological observations of the development of tumors have led to the concept that oncogenesis proceeds through the sequential acquisition of independent growth-related phenotypic characteristics (Foulds 1975). This process of tumor progression occurs through events involving the expression of specific genes affecting the regulation of cellular growth (recently reviewed in Weinberg 1985; Klein and Klein 1985; Bishop 1985). The requirement of two complementing oncogenes for the in vitro transformation of primary rat embryo firbroblasts provides a useful in vitro experimental model of tumor progression. Ha-ras (EJ) and v-myc or Ha-ras (T24) and E1A can act together to transform primary fibroblasts, while neither oncogene acting alone is capable of causing efficient transformation (Land et al. 1983; Ruley 1983) unless expressed at a high level (Spandidos and Wilkie 1984).

Chromosomally Integrated Retroviral Substrates are Sensitive Indicators of an Antibody Class Switch Recombinase-Like Activity

The main function of B lymphoid cells is to produce antibodies against an almost infinite variety of antigens which might be harmful to the organism. B cells comprise a clonally diverse population in which each cell has a mono-specific antibody receptor bound to its surface. A foreign antigen encounters its cognate antibody receptor bound to a B cell and, with the cooperation of helper T cells, causes the clonal proliferation and subsequent differentiation of this sub-set of B cells into antibody-secreting plasma cells [1].