Diana M. Lopez, Ph.D.

Professor of Microbiology and Immunology
Room 210, Papanicolaou Building
1550 NW 10th Avenue
Telephone: 305-243-6632
Fax: 305-243-4409
Email: dlopez@med.miami.edu

View Dr. Lopez's Molecular Immunobiology Course Manual

Research Interests:

A unique mucin immunoenhancing peptide with antitumor properties

Implantation of DA-3 mammary tumor cells into BALB/c mice results in tumor growth, metastatic lesions, and death. These cells were transfected with genes encoding for either the transmembrane (DA-3/TM) or secreted (DA-3/sec) form of human mucin 1 (MUC1). Although the gene for the secreted form lacks the transmembrane and cytoplasmic domains, the 5' sequences of these mucins are identical, however, the gene for the secreted mucin isoform ends with a sequence encoding for a unique eleven amino acid peptide. The DA-3/TM cells or DA-3 cells transfected with the neomycin vector only (DA-3/neo) have the same in vivo growth characteristics as the parent cell line. In contrast, DA-3/sec cells fail to grow when implanted in immunocompetent BALB/c animals. The mechanism of rejection was evaluated using mice deficient in constituents of the immune system. All mice lacking IFN-gamma, NK, NKT, or macrophages formed DA-3/sec tumors that regressed shortly after implantation. However, progressively growing DA-3/sec tumors developed in mice devoid of T lymphocytes. The importance of T lymphocytes in the rejection of DA-3/sec tumors was further supported by detection of DA-3 specific CTL in mice challenged with the DA-3/sec tumor. Recruitment of appropriate antigen presenting and effector cells is an important first step in the tumor clearance. Indeed, DA-3/sec cells or cell supernatants recruited 3-4 times as many macrophages as DA-3/TM cells in vivo, suggesting that a secreted chemotactic product is produced from DA-3/sec cells. RNA and protein analysis of DA-3/sec cells revealed that several genes are up-regulated by MUC1/sec expression, including monocyte chemoattractant protein 1 (CCL-2, also known as MCP-1). These results suggest DA-3/sec cells are capable of recruiting immune cells and that rejection of DA-3/sec tumors, while aided by cells of the innate immune response, is ultimately due to T cell mediated events.

Pre-exposure of BALB/c mice to DA-3/sec cells afforded protection against challenge with DA-3/TM or DA-3/neo mammary tumors and the unrelated tumors K7, an osteosarcoma, and RENCA, a renal cell carcinoma. Partial protection against subsequent tumor challenges was also achieved by substituting the 11 amino acid peptide found only in the secreted mucin-1 isoform, for the live DA-3/sec cells. Notably, the efficacy of this peptide is not strain restricted, as it also retarded the growth of Lewis lung carcinoma cells in C57 BL/6 mice. These findings reveal that a unique peptide present in the secreted MUC1 has immunoenhancing properties and may be a potential agent for use in immunotherapy.

Mammary tumor effects on thymic development and functions

The thymus is the major site of T-cell maturation; extensive proliferation, differentiation and apoptosis occur in this organ. During mammary tumorigenesis, there is a profound involution of the thymus associated with a severe depletion of the most abundant subset of thymocytes, CD4+8+ immature cells. Experiments to investigate the mechanism of loss of the CD4+8+ population indicated that there was no increase in the systemic levels of glucocorticoids, no loss of bone marrow precursors, and no decrease in precursor seeding of the thymus. Likewise, no enhanced emigration of thymocytes from the thymus to the periphery was observed in tumor bearing mice. A slight increase in apoptosis was found in tumor bearers = thymi, but there was no apparent decrease in the proliferation of early thymic precursors CD4-8- cells. Importantly, severely altered levels of subpopulations of the CD4-8- precursors consistent with an arrest in differentiation at an early stage of development were detected. Moreover, thymic stromal cell function appeared to become impaired during tumorigenesis, possibly due to the action of tumor-derived factors. Thus, downregulation of cell-mediated immune functions occurring at late stages of the disease may be causally related to the thymic involution occurring during mammary tumorigenesis.

Impaired functions of macrophages from tumor bearing mice

Peritoneal macrophages (PEM) from mammary tumor-bearing BALB/c mice (T-PEM) display a diminished ability to lyse tumor cells upon stimulation with LPS, a phenomenon that is associated to a lower production of nitric oxide (NO), and that is reverted upon costimulation with IFN-gamma. The reduced lytic activity and NO production displayed by T-PEM upon LPS activation were earlier shown to be due to a diminished transcription of the inducible nitric oxide synthase (iNOS) gene. We have investigated the participation of possible signaling molecules and transcription factors – PKC, NFKB, C/EBP and IRF-1 - in the downregulation of NO production in LPS-activated T-PEM. It was found that PKC activity was greatly reduced in T-PEM as compared to normal macrophages. Interestingly, the different PKC isozyme levels were not significantly altered in T-PEM, with the exception of PKC delta Alterations in the binding activity of the transcription factors NFKB and C/EBP appeared to be involved in the reduced transcription of iNOS previously observed in T-PEM after LPS activation. These results provide evidence that reductions in iNOS transcription secondary to alterations in cell signaling may be responsible for the diminished capacity of LPS-activated tumor-bearers’ macrophages to produce NO and lyse tumor targets.

Role of tumor associated factors in the upregulation of matrix metalloproteinase-9 (MMP-9) in T cells from tumor bearers

MMP-9, a matrix-degrading enzyme, is crucial in tumor invasion and metastasis and is implicated in leukocyte extravasation. During growth of the D1-DMBA-3 mammary tumor in BALB/c mice there is progressive upregulation of MMP-9 in splenic T-cells at both the transcriptional and translational levels. Our previous work has identified several factors produced by this tumor including PGE 2, GM-CSF, and phosphatidyl serine, however, none of these agents induces increased production of MMP-9 by normal splenic T cells. Although not produced by the tumor, TNF-α and IL-6 are upregulated in both macrophages and B cells in tumor bearing mice. Exposure of normal T cells to these two cytokines, however, also fails to upregulate MMP-9 production. Vascular endothelial growth factor (VEGF) is produced by many tumors and we determined that the mammary tumor used in our studies expresses high levels of this angiogenic growth factor. Importantly, splenic T cells from tumor bearers constitutively produce increased amounts of VEGF and treatment of normal T cells with VEGF results in upregulated MMP-9 production. Of crucial importance is the finding that tumor-infiltrating T cells also produce high levels of VEGF and MMP-9. Our studies indicate that VEGF can act directly on T lymphocytes and that elevated VEGF levels may contribute to the aberrant MMP-9 secretion by mammary tumor bearers’ T cells.

Selected Publications:

Adkins, B., Q.-L. Sun, V. Charyulu, D. Lobo, and D.M. Lopez. Early block in maturation Is associated with thymic involution in mammary tumor bearing mice. J. Immunol. 164:5635-5640 (2000).

Lopez, D.M., V. Charyulu, and B. Adkins. Influence of breast cancer on thymic function in mice. J. Mammary Gland Biology and Neoplasia 7:191-199 (2002).

Owen, J., V. Iragavarapu-Charyulu, Z. Gunja-Smith, L.M. Herbert, J.F. Grosso, and D.M. Lopez. Upregulation of matrix metalloproteinase 9 in T lymphocytes of mammary tumor bearers: Role of vascular endothelial growth factor. J. Immunol. 171:4340-4351 (2003).

Grosso, J.F., L.M. Herbert, J.L. Owen, and D.M. Lopez. MUC1/sec-expressing tumors are rejected in vivo by a T cell-dependent mechanism and secrete high levels of CCL2. J. Immunol. 173:1721-1730 (2004).

Herbert, L.M., J.F. Grosso, M. Dorsey Jr., I. Keydar, G. Kraus, M. Cejas, D. Wreschner, and D.M. Lopez. A unique mucin immunoenhancing peptide with antitumor properties. Cancer Research 64:8077-8084 (2004).

DiNapoli M.R., Torroella-Kouri M., Perry G., and Lopez D.M. Diminished PKC activity and decreased binding of transcription factors are involved in the impaired production of nitric oxide by macrophages from tumor-bearing mice. Int J Mol Med. 15(3):503-11 (2005).

Book Chapters:

Handel-Fernandez, M.E. and Lopez, D.M. Isolation of macrophages from tissues, fluids, and immune response sites. In: Macrophage Methodology: A practical approach (D.M. Paulnock, ed) Oxford University Press, Oxford. Chapter 1, pp. 1-30 (2000).

Lopez, D.M. Alterations of macrophage functions during mammary tumor development. In: Mechanisms of Tumor Escape from Immune Recognition (A. Ochoa, ed) Harwood Academic Publishers GMBH, Switzerland, Chapter 7, pp. 142-160 (2002).

Owen, J.L., V. Iragavarapu-Charyulu, and D.M. Lopez. T cell-derived matrix metalloproteinase-9 in breast cancer: friend or foe? In: Breast Disease. IOS Press, Amsterdam , the Netherlands 20:145-153 (2004).