Elizabeth Mushinski

Smad4 signalling in T cells is required for suppression of gastrointestinal cancer

SMAD4 (MAD homologue 4 (Drosophila)), also known as DPC4 (deleted in pancreatic cancer), is a tumour suppressor gene that encodes a central mediator of transforming growth factor-β signalling. Germline mutations in SMAD4 are found in over 50% of patients with familial juvenile polyposis, an autosomal dominant disorder characterized by predisposition to hamartomatous polyps and gastrointestinal cancer. Dense inflammatory cell infiltrates underlay grossly normal appearing, non-polypoid colonic and gastric mucosa of patients with familial juvenile polyposis. This prominent stromal component suggests that loss of SMAD4-dependent signalling in cells within the epithelial microenvironment has an important role in the evolution of intestinal tumorigenesis in this syndrome. Here we show that selective loss of Smad4-dependent signalling in T cells leads to spontaneous epithelial cancers throughout the gastrointestinal tract in mice, whereas epithelial-specific deletion of the Smad4 gene does not. Tumours arising within the colon, rectum, duodenum, stomach and oral cavity are stroma-rich with dense plasma cell infiltrates. Smad4-/- T cells produce abundant TH2-type cytokines including interleukin (IL)-5, IL-6 and IL-13, known mediators of plasma cell and stromal expansion. The results support the concept that cancer, as an outcome, reflects the loss of the normal communication between the cellular constituents of a given organ, and indicate that Smad4-deficient T cells ultimately send the wrong message to their stromal and epithelial neighbours.

AID-deficient Bcl-xL transgenic mice develop delayed atypical plasma cell tumors with unusual Ig/Myc chromosomal rearrangements

Activation-induced cytidine deaminase (AID) is required for immunoglobulin (Ig) class switch recombination and somatic hypermutation, and has also been implicated in translocations between Ig switch regions and c-Myc in plasma cell tumors in mice. We asked if AID is required for accelerated tumor development in pristane-treated Bcl-xL transgenic BALB/c mice deficient in AID (pBxAicda−/−). pBxAicda −/− mice developed tumors with a lower frequency (24 vs. 62%) and a longer mean latency (108 vs. 36 d) than AID-sufficient mice. The tumors appeared in oil granuloma tissue and did not form ascites. By interphase fluorescence in situ hybridization, six out of nine pBxAicda −/− primary tumors had T(12;15) and one had T(6;15) chromosomal translocations. Two tumors were transplantable and established as stable cell lines. Molecular and cytogenetic analyses showed that one had an unusual unbalanced T(12;15) translocation, with IgH Cμ and Pvt-1 oriented head to tail at the breakpoint, resulting in an elevated expression of c-Myc. In contrast, the second was T(12;15) negative, but had an elevated N-Myc expression caused by a paracentric inversion of chromosome 12. Thus, novel mechanisms juxtapose Ig and Myc-family genes in AID-deficient plasma cell tumors.

Immunoglobulin/Myc recombinations in Murine Peyer's patch follicles

Immunoglobulin heavy chain (Igh)/Myc recombinations are a hallmark of pristane‐induced mouse plasmacytomas but are also frequently found in non‐tumorous tissues. Here we describe for the first time a PCR‐based technique for detecting fusions between Ighμ or Ighα and Myc in situ. Igh/Myc recombinations were found in transplanted and primary plasmacytomas. In addition, the gut‐associated lymphoid tissues of plasmacytoma‐free BALB/c mice were investigated for the presence of Igh/Myc fusions. Igh/Myc rearrangements were detected in Peyers patch follicles and in the intestinal lamina propria both in normal mice and in mice shortly after pristane treatment. The sequence analysis showed that i) three to five different Igh/Myc hybrid sequences were present in individual follicles, ii) Igh/Myc recombinations can be subjected to additional switch recombinations as shown by related sequences in neighboring cells, and iii) cells harboring these rearrangements migrate into the adjacent lamina propria. The results indicate that Peyers patches are a hyper‐recombinogenic tissue. Myc recombination‐positive cells are present in at least 100‐fold more frequently than expected if recombinations were random, which suggests that this kind of trans‐chromosomal rearrangement may be targeted. Genes Chromosom. Cancer 20:1–8, 1997. Published 1997 Wiley‐Liss, Inc.

Clonal diversification of primary BALB/c plasmacytomas harboring T(12;15) chromosomal translocations

DNA sequence analysis of PCR amplified Igh/c-myc junction fragments of T(12;15) chromosome translocations and immunohistochemical determination of immunoglobulin isotype production were employed to study the clonal diversification of neoplastic translocated plasma cells that resided in peritoneal inflammatory granulomas of BALB/c mice harboring primary plasmacytomas. The diversity of plasma cells was found to take two major forms when the fine structure of the T(12;15) translocation was used as the clonotypic marker. First, mosaics of clones containing translocations that were apparently unrelated to each other were detected in nine out of 17 (53%) mice. Second, subclones derived from common T(12;15)+ progenitors by either secondary deletions in translocation breakpoint regions or aberrant isotype switching near translocation breaksites were found in five of 17 (29.5%) mice. When Ig expression was utilized as the clonotypic marker, clonal mosaics were shown to occur in all mice. This was demonstrated by the finding that the prevalent IgA- or IgG-producing plasmacytoma clone was invariably accompanied by smaller clones of IgG- or IgA-expressing neoplastic plasma cells, respectively. These results provided new insights into the clonal diversification at the terminal stage of plasmacytomagenesis. In addition, they suggested that BALB/c plasmacytomas may be uniquely useful for studying clonal diversity during B cell oncogenesis, since clonal evolution can be evaluated in a pool of tumor and tumor precursor cells that is clearly defined by the T(12;15) chromosomal translocation and the production of monoclonal immunoglobulin.

A new, mouse-myeloma immunoglobulin a having specificity for β-D(1→6)-linked D-galactopyranosyl residues

Abstract The discovery of T601, a new mouse-myeloma immunoglobulin A having specificity for β- D -(1→6)-linked D -galactopyranosyl residues, brings the total number of known antigalactan immunoglobulins to seven. The interaction of T601 with a number of ligands has been investigated. For those ligands showing interaction with the immunoglobulin, the affinity constants have been quantitatively measured by tryptophanyl fluorescence. The values show that protein T601 behaves very similarly to protein X24.

Cytotoxicity and Membrane Damage in vitro by Inclusion Complexes Between γ-Cyclodextrin and Siloxanes

Inclusion complexes of gamma-cyclodextrin and octamethylcyclotetrasiloxane (D4), decamethyltetrasiloxane (M10TS), and 1,3,5,7-tetramethyltetravinylcyclotetra - siloxane (TMTV-D4) were prepared to compare the cytotoxic effects of siloxanes in vitro. In these preparations, the hydrophobic siloxanes are surrounded by a hydrophilic shell of eight circularly linked D-glucose molecules (gamma-cyclodextrin), and upon contact with plasma membranes the siloxane molecule can intercalate into the lipid bilayer of the cell membrane. XRPC24, 2-11 plasmacytoma, CH12.LX lymphoma and P388D1 macrophage-like cells were used as indicator cells in toxicity assays. Using an MTT tetrazolium reduction to formazan test, a colorimetric method to determine the number of viable cells, the 50% minimal lethal doses (CD50) for the siloxane compounds were found to range from 30 to 50 microM. Sublethal doses (e.g., 15 microM and lower) resulted in the loss of lactate dehydrogenase (LDH) and glutathione (GSH) from the cytosolic compartment of the target cells and thus indicated cytotoxicity. Treatment of macrophages with siloxanes resulted in a higher production of interleukin-6 (IL-6) than was exhibited by untreated macrophages. The B9 cell bioassay of these treated cells showed as much as a 10 fold higher production (500 U/ml) of IL-6 than did the untreated cells. The degree of increase was dependent on the compound and concentration used. The results of this study show that low molecular weight siloxanes produce lethal effects on B-lymphocyte derived target cells in vitro and permeabilize the plasma membranes at lower sublethal concentrations.

Rapid Induction of Plasmacytomas in Mice by Pristane and a Murine Recombinant Retrovirus Containing an Avian v-myc and a Defective raf Oncogene

Plasma cell tumors can be induced in genetically susceptible BALB/cAn mice by the intraperitoneal injection of various kinds of mineral (paraffin) oils or available components of these oils. Currently, the most widely used agent is pristane 2,6,10,14 tetramethy1pentadecane. The incidence of plasma cell tumors obtained in BALB/cAn mice varies according to the dose of pristane. The highest incidence of plasmacytomas occurs when mice are given three 0.5 ml i. p. injections of pristane, spaced 2 months apart (Potter and Wax 1983). Approximately 60% of the mice treated with this regimen develop plasmacytomas with a minimal latent period of 120 days and a mean latent period of 210–220 days. In contrast, when mice are given a single injection of 0.5 ml pristane i. p., only 25–30% develop plasmacytomas. The minimal latent period is 140 days and the mean latent period ca. 215 days.

Generation of Immunoglobulin/c-myc Recombinations in Murine Peyer’s Patch Follicles

Plasmacytoma can be induced by the intraperitoneal injection of pristane in susceptible BALB/c mice. Most inbred strains of mice, however, are resistant to this type of plasmacytoma induction (Potter & Wiener, 1992).

Characterization of idiotypes on antibodies to galactan.

The correlation between the primary structure and the expression of idiotypy in the anti-galactan antibody system is extremely precise. Amino-acid residues, which are implicated in the expression of recurrent or private idiotopes, are localized in the CDR3 region of all hybridomas and of at least 2 out of 4 myeloma proteins. A single substitution in this region may, in some cases, suffice to abolish idiotope expression.

Induced Plasmacytoma Formation in Mice

Although plasmacytomas can develop spontaneously in mice, the more common origin requires inducing agents. In the system which we will describe, plasmacytomagenesis is initiated by the intraperitoneal injection of paraffin oils such as the alkane pristane (2,6,10,14-tetramethylpentadecane) (Anderson and Potter 1969). These tumors can also be induced by the intraperitoneal implantation of solid plastic materials as was originally discovered by Merwin and Algire (see Merwin et al. 1963). An important characteristic of plasmacytoma induction in mice is that it only works in certain inbred strains such as BALB/cAn in which the expected average incidence is ∼ 60%, or NZB where between 30 and 40% of the mice develop these tumors; most other inbred strains do not develop these tumors (Morse et al. 1980). Plasmacytomas have been studied cytogentically by Francis Wiener and Shinsuke Ohno, and over 95% of them have reciprocal chromosomal translocations involving immunoglobulin genes (chromosomes 12 and 6 and 16) and c-myc (chromosome 15) or loci near c-myc such as Pvt-1 (Ohno et al. 1979,1984). These translocations cause deregulation of c-mvc transcription (Mushinski 1988). Deregulation of c-myc cooperates with other oncogenic mutations to transform certain B-lymphocytes but does not appear to be sufficient by itself to cause transformation (see Potter 1990). C-myc deregulation is consistently found in spontaneous rat immunocytomas (plasma cell tumors) and in Burkitt’s lymphoma in man (see Potter 1990).