Deborah A. Lebman

The role of TGF-β in growth, differentiation, and maturation of B lymphocytes

Transforming growth factor-β (TGF-β) affects B cells at all stages in development. It appears to be involved in lymphopoiesis and is required for the development of plasma cells secreting all secondary isotypes. Its ability to inhibit proliferation and stimulate apoptosis suggest that it may be involved both in germinal center development and regulation of B-cell proliferation at sites of high antigen load such as the gastrointestinal tract. Although TGF-β appears to be required for the generation of B cells secreting secondary isotypes, it inhibits secretion of IgM and IgA from cells expressing those isotypes. In this regard, TGF-β may alter the level of RNA processing factors either directly or indirectly by inhibiting progression through the cell cycle. One of the best characterized effects of TGF-β is its ability to stimulate isotype switching to IgA in both mouse and man. There is some controversy concerning its mechanism of action in this process, but its critical role is without question. The controversy may stem in part from an inability to separate the effects of endogenous and exogenous TGF-β in the multiple models of isotype switching. The influence of endogenous TGF-β is perhaps best exemplified by analysis of production of the different classes of IgG in mouse strains producing different levels of TGF-β.

Inability of Transforming Growth Factor-β to Cause SnoN Degradation Leads to Resistance to Transforming Growth Factor-β–Induced Growth Arrest in Esophageal Cancer Cells

It is well established that loss of a growth inhibitory response to transforming growth factor-beta (TGF-beta) is a common feature of epithelial cancers including esophageal cancer. However, the molecular basis for the abrogation of this key homeostatic mechanism is poorly understood. In esophageal cancer cell lines that are resistant to TGF-beta-induced growth inhibition, TGF-beta also fails to decrease transcription of c-myc despite the presence of functional signaling components. Consequently, to gain a better understanding of the mechanisms leading to resistance to TGF-beta-induced growth arrest, the basis for the inability to decrease c-myc transcription was investigated. Regardless of sensitivity to TGF-beta-induced growth arrest, TGF-beta enhanced the ability of Smad3-protein complexes to bind c-myc regulatory elements. However, in a growth inhibition-resistant esophageal cancer cell line, the Smad3-protein complexes contained the SnoN oncoprotein. Furthermore, in esophageal cancer cell lines that are resistant to TGF-beta-induced growth arrest, TGF-beta does not cause degradation of SnoN. Analyses of the effect of modulating SnoN expression in both growth inhibition-sensitive and growth inhibition-resistant cell lines showed that degradation of SnoN is a prerequisite for both TGF-beta-induced repression of c-myc transcription and growth arrest. The data indicate that SnoN-Smad3 complexes do not cause repression of c-myc transcription but rather prevent functionality of active repressor complexes. Thus, these studies reveal a novel mechanism for resistance to TGF-beta-induced growth inhibition in esophageal cancer, namely the failure to degrade SnoN. In addition, they show that SnoN can block TGF-beta repression of gene transcription.

Photocaged permeability: a new strategy for controlled drug release

Light is used to release a drug from a cell impermeable small molecule, uncloaking its cytotoxic effect on cancer cells.

The Microenvironment of Human Peyer’s Patches Inhibits the Increase in CD38 Expression Associated with the Germinal Center Reaction

Analysis of B cells in the human tonsils identified CD38 expression as a hallmark of germinal center (GC) B cells. However, the signals responsible for the in vivo induction of CD38 have not been determined. The primary site for generation of memory and plasma cells in the gastrointestinal tract is the GCs of Peyer’s patches (PP). PP and intestinal mucosa, but not tonsils or oral mucosa, express mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The ligand for MAdCAM-1, integrin α4β7, is expressed on naive B cells and memory B cells that traffic to the gastrointestinal tract. In this study we determine that, unlike tonsil, human PP GC B cells do not express significant levels of CD38. PP B cells can be induced to express CD38 upon culture with CD40 ligand, anti-B cell receptor, and IFN-γ. However, coculture of tonsil naive B cells with an Ab directed against integrin β7 inhibits IFN-γ-induced CD38 hyperexpression. The absence of CD38 on PP GCs suggests that there are tissue-specific pathways of B cell development that differ between tonsil and PP. The differential expression pattern of MAdCAM-1, together with the observation that ligation of β7 can inhibit the induction of CD38 expression, suggests that ligation of α4β7 in vivo may contribute to a PP-specific GC phenotype.

Correct Immunoglobulin α mRNA Processing Depends on Specific Sequence in the Cα3-αM Intron

The maturation of IgM-expressing B cells to IgM-secreting plasma cells is associated with both an increase in μ mRNA and the ratio of secreted to membrane forms of μ mRNA which differ at the 3′ termini. In contrast, both in vitro and in vivo the secreted form of α mRNA is predominant at all stages in the development of a secretory IgA response. Previous studies demonstrated that preferential usage of the αs poly(A) site does not result from transcription termination and is independent of either the poly(A) sites or the 3′ splice site associated with the exon encoding the membrane exon of IgA (αM). The present study demonstrates that a 349-bp region located 774 bp 3′ to the αs poly(A) site is required for the preferential usage of the αs terminus. This region, which is the first isotype-specific cis-acting regulatory sequence not immediately adjacent to a secretory poly(A) site to be identified, contains regulatory elements that increase the efficiency of polyadenylation/cleavage. A ubiquitous, ∼58-kDa RNA-binding protein interacts specifically with this regulatory region. These studies support the premise that cis-acting elements unique to each CH gene can impinge upon a common mechanism regulating Ig mRNA processing.

DEVELOPMENTAL REGULATION OF IMMUNOGLOBULIN MRNA PROCESSING AND THE IGA RESPONSE : ESTABLISHING A PARADIGM

IgA, which is protective at mucosal sites, is derived from memory B cells that develop in the organized lymphoid tissue of the gastrointestinal tract and subsequently mature to plasma cells in the lamina propria. Similarly to B cells expressing other isotypes, the maturation of IgA-expressing B cells is associated with both an increase in the steady-state level of immunoglobulin mRNA and the ratio of secreted to membrane forms of mRNA, which differ in 3′ terminus. In contrast to B cells expressing other isotypes, at all stages in the development of an IgA response, the secreted form of α mRNA predominates. In this article, studies on the general features of IgA B cell development, mechanisms regulating 3′ terminus usage of Ig mRNAs, and isotype-specific regulation of 3′ terminus usage particularly in regard to α mRNA are discussed.

Abstract 5665: New alternatives to photodynamic therapy based on light-enabled drug delivery.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Conventional photodynamic therapy (PDT) aims to achieve tumor drug targeting by selectively killing cells upon illumination. PDT serves as a non-invasive treatment option for many types of non-metastatic, light-accessible cancers such as those involving the head and neck, lung, bladder, prostate, or esophagus. The various types of PDT all use the same basic strategy for killing cells; photosensitizing molecules are taken up by cancer cells, and, upon illumination, singlet oxygen is generated in the vicinity of the photosensitizer, killing the cell. A new alternative to PDT is photochemical internalization, in which singlet oxygen generated by a photosensitizer disrupts endosomes containing a toxin, releasing it into the cytoplasm and killing the cell. A drawback to both of these approaches is that moderate levels of oxygen are required, yet tumors are often hypoxic environments. Moreover, during PDT treatment, oxygen levels drop even further as the singlet oxygen is consumed; this can compromise treatment effectiveness. We have sought to address these challenges by developing new strategies for light-based treatment of cancer that are oxygen independent. In the first approach, which we call photocaged permeability, we have attached the anticancer drug doxorubicin to a cell impermeable small molecule via a light-cleavable linker. We show using flow cytometry and confocal microscopy that this conjugate is cell impermeable in the dark, yet upon illumination, doxorubicin is released and enters the cells. The IC50 for our conjugate in JH-EsoAd1 cells using non-cytotoxic doses of light is 1.6 μM, identical to that of doxorubicin alone. No cytotoxicity is observed at doses 30-fold higher in the dark. In a second approach, we have attached doxorubicin to folate receptor and PSMA ligands via a light cleavable linker in order to enable dual cancer cell targeting with both light and ligand specificity. The ability of the resulting conjugates to selectively kill cells expressing the requisite receptors with light will be described. Citation Format: M. Michael Dcona, Deboleena Mitra, Rachel W. Goehe, David A. Gewirtz, Deborah A. Lebman, Matthew C.T. Hartman. New alternatives to photodynamic therapy based on light-enabled drug delivery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5665. doi:10.1158/1538-7445.AM2013-5665