Enza Ferrero

Evolution and Function of the ADP Ribosyl Cyclase/CD38 Gene Family in Physiology and Pathology

The membrane proteins CD38 and CD157 belong to an evolutionarily conserved family of enzymes that play crucial roles in human physiology. Expressed in distinct patterns in most tissues, CD38 (and CD157) cleaves NAD(+) and NADP(+), generating cyclic ADP ribose (cADPR), NAADP, and ADPR. These reaction products are essential for the regulation of intracellular Ca(2+), the most ancient and universal cell signaling system. The entire family of enzymes controls complex processes, including egg fertilization, cell activation and proliferation, muscle contraction, hormone secretion, and immune responses. Over the course of evolution, the molecules have developed the ability to interact laterally and frontally with other surface proteins and have acquired receptor-like features. As detailed in this review, the loss of CD38 function is associated with impaired immune responses, metabolic disturbances, and behavioral modifications in mice. CD38 is a powerful disease marker for human leukemias and myelomas, is directly involved in the pathogenesis and outcome of human immunodeficiency virus infection and chronic lymphocytic leukemia, and controls insulin release and the development of diabetes. Here, the data concerning diseases are examined in view of potential clinical applications in diagnosis, prognosis, and therapy. The concluding remarks try to frame all of the currently available information within a unified working model that takes into account both the enzymatic and receptorial functions of the molecules.

The metamorphosis of a molecule: from soluble enzyme to the leukocyte receptor CD38

Human CD38 is a 45‐kDa type II membrane glycoprotein with an intricate pattern of expression in leukocytes, although evidence is accumulating of its quite widespread expression in cells of nonvascular origin. CD38 is a member of a nascent eukaryotic gene family encoding cytosolic and membrane‐bound enzymes whose substrate is NAD, a coenzyme ubiquitously distributed in nature. Functionally, CD38 is an eclectic molecule with the ability not only to catalyze but also to signal, to mobilize calcium, and to adhere to itself, to hyaluronan, and to other ligands. Interaction with CD38 on various leukocyte subpopulations has profound though diverse consequences on their life‐span, but these effects seem to be independent of the enzymatic activity of the molecule. CD38 challenges our expectations of a surface molecule and we must sift through its many guises to unmask its true nature. J. Leukoc. Biol. 65: 151–161; 1999.

CD38 and CD157: A long journey from activation markers to multifunctional molecules

CD38 (also known as T10) was identified in the late 1970s in the course of pioneering work carried out at the Dana‐Farber Cancer Center (Boston, MA) that focused on the identification of surface molecules involved in antigen recognition. CD38 was initially found on thymocytes and T lymphocytes, but today we know that the molecule is found throughout the immune system, although its expression levels vary. Because of this, CD38 was considered an “activation marker,” a term still popular in routine flow cytometry. This review summarizes the findings obtained from different approaches, which led to CD38 being re‐defined as a multifunctional molecule. CD38 and its homologue CD157 (BST‐1), contiguous gene duplicates on human chromosome 4 (4p15), are part of a gene family encoding products that modulate the social life of cells by means of bidirectional signals. Both CD38 and CD157 play dual roles as receptors and ectoenzymes, endowed with complex activities related to signaling and cell homeostasis. The structure‐function analysis presented here is intended to give clinical scientists and flow cytometrists a background knowledge of these molecules. The link between CD38/CD157 and human diseases will be explored here in the context of chronic lymphocytic leukemia, myeloma and ovarian carcinoma, although other disease associations are also known. Thus CD38 and CD157 have evolved from simple leukocyte activation markers to multifunctional molecules involved in health and disease. Future tasks will be to explore their potential as targets for in vivo therapeutic interventions and as regulators of the immune response.

The human CD38 gene: polymorphism, CpG island, and linkage to the CD157 (BST-1) gene.

Abstract CD38 is a leukocyte activation antigen and ectoenzyme [NAD(P)+ glycohydrolase; EC 3.2.2.6] involved in numerous immune functions. The human CD38 gene is complex [eight exons, >80 kilobases (kb) long] located on Chromosome 4p15, and part of the eukaryotic NAD+ glycohydrolase/ADP-ribosyl cyclase gene family. Because of the increasing relevance of the CD38 molecule in the host immune response to infectious, tumoral, and metabolic diseases, we investigated the genetic variability and linkage of the human CD38 locus. We report that (1) the restriction endonuclease Pvu II identifies a bi-allelic polymorphism here defined as formed by the alleles CD38*A (12 kb) and CD38*B (9/2.5 kb); (2) their frequency in the healthy Italian Caucasian population is 14% and 86%, respectively; (3) the polymorphic Pvu II site is located at the 5′ end of the first intron of the CD38 gene; (4) in conjunction with the polymorphic site, we identified a 900 base pair CpG island associated with the CD38 gene, with two potential Sp1 binding sites; (5) the CpG island may play a role in the regulation of CD38 expression and is hypomethylated in various cell lines; (6) by pulsed-field gel electrophoresis we show that CD38 and its paralogue, the bone-marrow stromal cell antigen BST-1 (CD157), map to the same 800 kb Avi II fragment, indicating that the two human ecto-NADase genes are closely linked.

CD38 is expressed on human mature monocyte-derived dendritic cells and is functionally involved in CD83 expression and IL-12 induction

Dendritic cell (DC) maturation is characterized by the gain or loss of immunological functions and by expression of distinctive surface receptors. CD38 is an ectoenzyme that catalyzes the synthesis of cyclic ADP ribose (a potent second messenger for Ca2+ release), as well as a receptor that initiates transmembrane signaling upon engagement with its counter‐receptor CD31 or with agonistic monoclonal antibodies. Since CD38 is expressed by resting monocytes, we aimed to monitor CD38 expression during the differentiation of human monocyte‐derived DC (MDDC) and to investigate the possibility that CD38 plays a functional role during DC maturation. CD38 is down‐modulated during differentiation into immature MDDC and expressed again upon maturation. The extent of CD38 expression is dependent on the stimulus adopted (LPS > IFN‐γ > CD40 cross‐linking). Although weak, IFN‐γ consistently induces DC maturation. De novo‐synthesized CD38 is enzymatically active, and its expression in mature (m) MDDC is dependent on NF‐κB activity. However, CD38 is not merely a maturation marker but also mediates signaling in mMDDC, where it maintains its functions as a receptor. Activation via agonistic anti‐CD38 mAb induces up‐regulation of CD83 expression and IL‐12 secretion, whereas disruption of CD38/CD31 interaction inhibits CD83 expression, IL‐12 secretion and MDDC‐induced allogeneic T cell proliferation.

Arsenic Trioxide and Breast Cancer: Analysis of the Apoptotic, Differentiative and Immunomodulatory Effects

Arsenic trioxide (As2O3) is used clinically to treat acute promyelocytic leukemia and has activity in vitro against several solid tumour cell lines, where induction of differentiation and apoptosis are the prime effects. To investigate the potential therapeutic application of As2O3 to breast cancer, we analysed the effects of As2O3 on the growth of four human breast cancer cell lines: MCF7, MDA-MB-231, T-47D and BT-20. Cells were cultured in 0.5, 2 and 5 μM As2O3, a range of pharmacologically achievable concentrations of As2O3. At ≥2 μM, As2O3 rapidly induced cell death by apoptosis in MCF7 and MDA-MB-231 while T-47D and BT-20 were partially resistant. At 0.5 μM, As2O3 was subapoptotic but induced features of differentiation consisting in upregulation of ICAM-1 (CD54), a marker of mammary epithelial differentiation, and cell cultures appeared morphologically more orga-nized. Furthermore, we demonstrate by standard cytotoxicity assays that As2O3 treatment can augment breast cancer cell lysis by lymphokine-activated killer cells and demonstrate an important role of the ICAM-1/LFA-1 interaction in this process. This additional activity of As2O3 could translate into improved antitumour immunosurveillance in vivo. In conclusion, As2O3 induced varying degrees of differentiation, apoptosis and lysis in these model cell lines, and may be a promising adjuvant to current treatments of breast cancer by virtue of its triple apoptotic, differentiative and immunomodulatory effects.

CD38 gene polymorphism and chronic lymphocytic leukemia: a role in transformation to Richter syndrome?

CD38 rules proliferation signals in chronic lymphocytic leukemia (CLL) cells, suggesting that the molecule is not merely a prognostic marker but also a key element in the pathogenetic network underlying the disease. CD38 has a genetic polymorphism, characterized by a C>G variation in the regulatory region of intron 1. The working hypothesis is that the presence of different alleles in CLL patients marks (or accounts for) some of the clinical heterogeneity. CD38 allele distribution in 248 Italian patients overlapped with that of the controls (n = 232), suggesting that susceptibility to CLL is not influenced by CD38 genotype. Stratification of patients according to markers of unfavorable prognosis constantly resulted in a significantly higher frequency of the rare G allele. Furthermore, analysis of clinical parameters showed that G allele is independently associated with nodal/splenic involvement. The highest G allele frequency was observed in the 16 patients of the cohort that developed Richter syndrome (RS). Five-year cumulative incidence of transformation was significantly higher in G allele carriers than in CC homozygotes. Multivariate analysis on a total of 30 RS patients confirmed that the probability of transformation is strongly associated with G allele, likely representing an independent risk factor for RS development.

A large genomic deletion leads to enhancer adoption by the lamin B1 gene: a second path to autosomal dominant adult-onset demyelinating leukodystrophy (ADLD).

Chromosomal rearrangements with duplication of the lamin B1 (LMNB1) gene underlie autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), a rare neurological disorder in which overexpression of LMNB1 causes progressive central nervous system demyelination. However, we previously reported an ADLD family (ADLD-1-TO) without evidence of duplication or other mutation in LMNB1 despite linkage to the LMNB1 locus and lamin B1 overexpression. By custom array-CGH, we further investigated this family and report here that patients carry a large (∼660 kb) heterozygous deletion that begins 66 kb upstream of the LMNB1 promoter. Lamin B1 overexpression was confirmed in further ADLD-1-TO tissues and in a postmortem brain sample, where lamin B1 was increased in the frontal lobe. Through parallel studies, we investigated both loss of genetic material and chromosomal rearrangement as possible causes of LMNB1 overexpression, and found that ADLD-1-TO plausibly results from an enhancer adoption mechanism. The deletion eliminates a genome topological domain boundary, allowing normally forbidden interactions between at least three forebrain-directed enhancers and the LMNB1 promoter, in line with the observed mainly cerebral localization of lamin B1 overexpression and myelin degeneration. This second route to LMNB1 overexpression and ADLD is a new example of the relevance of regulatory landscape modifications in determining Mendelian phenotypes.

Estrogen Receptor α Is a Novel Marker Expressed by Follicular Dendritic Cells in Lymph Nodes and Tumor-Associated Lymphoid Infiltrates

During routine assessment of the hormonal phenotype of breast carcinomas, we detected expression of the estrogen receptor (ER) in the germinal centers of reactive lymphoid follicles surrounding malignant foci. To confirm and extend this finding, we compared ER-alpha, progesterone receptor (PR), and androgen receptor (AR) immunostaining in hyperplastic or metastatic lymph nodes obtained from patients with various pathology, disease location, gender and age. Irrespective of these parameters, we found that: 1) ER-alpha-positive cells were located prevalently in germinal centers, 2) the PR was weakly expressed by cells within and surrounding germinal centers, and 3) the androgen receptor was undetectable. Transcripts for ER-alpha and PR were also detected by reverse transcription-polymerase chain reaction on laser-microdissected lymph node germinal centers. Morphologically, the ER-positive cells resemble dendritic cells and by double immunostaining were found to express both CD21 and CD23, which is characteristic of follicular dendritic cells. Finally, we assessed the effects of Tamoxifen treatment by comparing the numbers of ER-positive follicular dendritic cells in lymph nodes obtained from breast cancer patients before and after treatment. The results show that Tamoxifen treatment generated larger germinal centers with more abundant ER(+)/CD21(+)/CD23(+) cells. Taken together, these results open new perspectives on the effects of sex steroids and their antagonists on the human response in cancer and inflammation.

Functional Role and Prognostic Significance of CD157 in Ovarian Carcinoma

BACKGROUND CD157, an ADP-ribosyl cyclase-related cell surface molecule, regulates leukocyte diapedesis during inflammation. Because CD157 is expressed in mesothelial cells and diapedesis resembles tumor cell migration, we investigated the role of CD157 in ovarian carcinoma. METHODS We assayed surgically obtained ovarian cancer and mesothelial cells and both native and engineered ovarian cancer cell lines for CD157 expression using flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR), and for adhesion to extracellular matrices, migration, and invasion using cell-based assays. We investigated invasion of human peritoneal mesothelial cells by serous ovarian cancer cells with a three-dimensional coculture model. Experiments were performed with or without CD157-blocking antibodies. CD157 expression in tissue sections from ovarian cancer patients (n = 88) was examined by immunohistochemistry, quantified by histological score (H score), and categorized as at or above or below the median value of 60, and compared with clinical parameters. Statistical tests were two-sided. RESULTS CD157 was expressed by ovarian cancer cells and mesothelium, and it potentiated the adhesion, migration, and invasion of serous ovarian cancer cells through different extracellular matrices. CD157-transfected ovarian cancer cells migrated twice as much as CD157-negative control cells (P = .001). Blockage of CD157 inhibited mesothelial invasion by serous ovarian cancer cells in a three-dimensional model. CD157 was expressed in 82 (93%) of the 88 epithelial ovarian cancer tissue specimens. In serous ovarian cancer, patients with CD157 H scores of 60 or greater had statistically significantly shorter disease-free survival and overall survival than patients with lower CD157 H scores (CD157 H score > or =60 vs <60: median disease-free survival = 18 months, 95% confidence interval [CI] = 5.92 to 30.07 vs unreached, P = .005; CD157 H score > or =60 vs <60: median overall survival = 45 months, 95% CI = 21.21 to 68.79 vs unreached, P = .024). Multivariable Cox regression showed that CD157 is an independent prognostic factor for recurrence (hazard ratio of disease recurrence = 3.01, 95% CI = 1.35 to 6.70, P = .007) and survival (hazard ratio of survival = 3.44, 95% CI = 1.27 to 9.31, P = .015). CONCLUSIONS CD157 plays a pivotal role in the control of ovarian cancer cell migration and peritoneal invasion, and it may be clinically useful as a prognostic tool and therapeutic target.