Alvin M. Malkinson

Molecular Comparison of Human and Mouse Pulmonary Adenocarcinomas

Mice develop lung tumors similar in their histogenesis and molecular features to peripheral adenocarcinomas in humans. The advantage of this model system is that events early in tumorigenesis can be delineated and their biological consequences tested by transgenic and knockout strategies. Both human and murine adenocarcinomas contain Kras mutations; in mice these occur within weeks following carcinogen administration. Decreased expression of similar tumor suppressor genes occur in both species due to mutation, deletion, altered DNA methylation, or unknown mechanisms. These genes include p15, p16, Rb, cyclin D1, p53, Apc, Mcc, and Gjal. Some genes have only been examined in one of these species, such as the deletions in chromosome 3p and the overexpression of bcl 2 in human adenocarcinoma. Not all molecular changes are identical to the two species, however. Quinone oxidoreductase (DT-diaphorase) levels rise in the human tumors but fall in the mouse; the extent of both changes is very dramatic. Similarly, EGF-receptor content often increases in human lung adenocarcinomas but decreases in the mouse tumors. In general, however, the nature of the molecular changes is quite similar.

The genetic basis of susceptibility to lung tumors in mice

This is the first in a series of review articles describing the current state of research on mouse lung tumorigenesis. The system is valuable as a biological model for studying stages of tumor development and the interaction of genetic and environmental factors which dispose towards neoplasia. Additionally, these tumors are analagous to bronchiolo-alveolar cancer in man. Three pulmonary adenoma susceptibility (Pas) genes regulate susceptibility; 1 of these is the proto-oncogene, K-ras2. Candidates for the other 2 genes include the H-2 histocompatibility locus and genes which regulate the basal proliferative rate of the cells from which these tumors arise. Tumor development is favored by a depressed immune system, immature age, and decreased levels of circulating corticosterone.

Calpain and calpastatin regulate neutrophil apoptosis

The average polymorphonuclear neutrophil (PMN) lives only a day and then dies by apoptosis. We previously found that the calcium‐dependent protease calpain is required for apoptosis in several mouse models of cell death. Here we identify calpain, and its endogenous inhibitor calpastatin, as regulators of human neutrophil apoptosis. Cell death triggered by the translation inhibitor cycloheximide is calpain‐dependent, as evidenced using either a calpain active site inhibitor (N‐acetyl‐leucyl‐leucyl‐norleucinal) or agents that target calpains calcium binding sites (PD150606, PD151746). No significant effect on cycloheximide‐triggered apoptosis was found by using inhibitors of the proteasome or of other papain‐like cysteine proteases, providing further evidence that the active site calpain inhibitor prevents apoptosis via its action on calpain. In addition, we find that potentiation of calpain activity by depleting its endogenous inhibitor, calpastatin, is sufficient to cause apoptosis of neutrophils. Nevertheless, apoptosis signalled via the Fas antigen proceeds regardless of the presence of calpain inhibitor. These experiments support a growing body of work, indicating an upstream regulatory role for calpain in many, but not all, forms of apoptotic cell death. They also identify calpastatin as a participant in apoptotic cell death and suggest that for at least one cell type, a decrease in calpastatin is a sufficient stimulus to initiate calpain‐dependent apoptosis. J. Cell. Physiol. 178:311–319, 1999.

Mouse lung epithelial cell lines--tools for the study of differentiation and the neoplastic phenotype.

Several dozen lung epithelial cell lines have been established in culture over the past 20 years from normal lung explants and their spontaneous transformants, and from lung tumors that arose spontaneously or were induced with chemicals, viruses, or oncogenic transgenes. To provide information from which to choose appropriate lines for investigating problems in lung cell biology and pulmonary neoplasia, this review describes the origins of these lines and some of their characteristics. These include growth, morphology, tumorigenicity, ability to metastasize, xenobiotic metabolism, mutational status, signal transducing activities, cytogenetics, ability to form domes, and electric conductance. In addition to collecting this information in a single place for the first time, we describe previously unpublished apoptosis features of some of these lines. An increasing number of investigations are beginning to use these lines and this review contains references into 1997.

Clara cell: progenitor for the bronchiolar epithelium.

Clara cells were first described as a morphologically distinct cell type by Kolliker in 1881, but they take their name from the seminal study of human and rabbit bronchioles by Max Clara in 1937. Since their discovery, Clara cells have been identified as central players in protecting the airway from environmental exposures. The diverse functions of Clara cells in lung homeostasis include roles in xenobiotic metabolism, immune system regulation, and progenitor cell activity. Recent identification of a sub-population of Clara cells as a bronchiolar tissue-specific stem cell and a potential tumor initiating cell has focused the attention of cell and molecular biologists on the Clara cell and its behavior under normal and disease conditions.

Primary lung tumors in mice as an aid for understanding, preventing, and treating human adenocarcinoma of the lung

Primary lung tumors in mice have morphologic, histogenic, and molecular features similar to human lung adenocarcinoma, and in particular, the bronchiolo-alveolar carcinoma subtype. Because of this, and because of the genetic homology between man and mouse and the ease of genetic manipulations in mice, this model system is receiving intense research attention. This review is intended to be informative to clinical investigators, and describes features of this model, how it is being used for translational research, and points out additional avenues of study that could have practical benefits, such as application for identifying novel therapeutic strategies.

Differential Expression and Localization of the mRNA Binding Proteins, AU‐Rich Element mRNA Binding Protein (AUF1) and Hu Antigen R (HuR), in Neoplastic Lung Tissue

Modulation of gene expression at the level of mRNA stability has emerged as an important regulatory paradigm. In this context, differential expression of numerous mRNAs in normal versus neoplastic tissues has been described. Altered expression of these genes, at least in part, has been demonstrated to be at the level of mRNA stability. Two ubiquitously expressed mRNA binding proteins have recently been implicated in the stabilization (Hu antigen R/HuR) or destabilization (AU‐rich element mRNA binding protein [AUF1]/heterogeneous nuclear ribonucleoprotein D) of target mRNAs. Further, their functional activity appears to require cytoplasmic localization. In the present study, we demonstrate a strong correlation between increased cytoplasmic expression of both AUF1 and HuR with urethane‐induced neoplasia and with butylated hydroxytoluene–induced compensatory hyperplasia in mouse lung tissue. In addition, when compared with slower growing cells, rapidly growing neoplastic lung epithelial cell lines expressed a consistently higher abundance of both AUF1 and HuR proteins. Moreover, in nontumorigenic cell lines, both AUF1 and HuR protein abundance decreased with confluence and growth arrest. In contrast, in spontaneous transformants, AUF1 and HuR abundance was unaffected by changes in cell density. We suggest that growth‐regulated alterations in AUF1 and HuR abundance may have pleiotropic effects on the expression of a number of highly regulated mRNAs and that this significantly impacts the onset, maintenance, and progression of the neoplastic phenotype. Mol. Carcinog. 28:76–83, 2000.

Ki-ras and p53 Mutations Are Early and Late Events, Respectively, in Urethane-Induced Pulmonary Carcinogenesis in A/J Mice.

In the A/J strain of mice, urethane (ethyl carbamate) induces lung hyperplasia, adenoma, and adenocarcinoma in a time‐dependent manner. These distinct morphological stages may correlate with sequential molecular genetic changes in this mouse model. To test this hypothesis, we investigated the presence of mutations involving Ki‐ras and p53 in urethane‐induced lung lesions in A/J mice at early and late stages of tumorigenesis. We precisely microdissected 40 lung lesions from paraffin‐embedded sections. Ki‐ras mutations around codon 61 and p53 mutations in exons 5–8 were identified by polymerase chain reaction‐single‐strand conformation polymorphism and DNA sequencing techniques. In 29 early‐stage lung lesions classified as hyperplasias (seven) or adenomas (22), we observed 19 Ki‐ras mutations (66%), including three silent mutations and one double mutation at different codons, and one silent p53 mutation (3.5%). In 11 late‐stage adenomas, we identified nine activating Ki‐ras mutations (82%) and four missense p53 mutations (36%). These results indicate that Ki‐ras mutations arise early, whereas p53 mutations occur relatively late during the benign stages of urethane‐induced lung carcinogenesis in A/J mice.

Growth inhibition in G1 and altered expression of cyclin D1 and p27kip-1after forced connexin expression in lung and liver carcinoma cells

Gap junctional intercellular communication (GJIC) and connexin expression are frequently decreased in neoplasia and may contribute to defective growth control and loss of differentiated functions. GJIC, in E9 mouse lung carcinoma cells and WB‐aB1 neoplastic rat liver epithelial cells, was elevated by forced expression of the gap junction proteins, connexin43 (Cx43) and connexin32 (Cx32), respectively. Transfection of Cx43 into E9 cells increased fluorescent dye‐coupling in the transfected clones, E9‐2 and E9‐3, to levels comparable to the nontransformed sibling cell line, E10, from which E9 cells originated. Transduction of Cx32 into WB‐aB1 cells also increased dye‐coupling in the clone, WB‐a/32‐10, to a level that was comparable to the nontransformed sibling cell line, WB‐F344. The cell cycle distribution was also affected as a result of forced connexin expression. The percentage of cells in G1‐phase increased and the percentage in S‐phase decreased in E9‐2 and WB‐a/32‐10 cells as compared to E9 and WB‐aB1 cells. Concomitantly, these cells exhibited changes in G1‐phase cell cycle regulators. E9‐2 and WB‐a/32‐10 cells expressed significantly less cyclin D1 and more p27kip‐1 protein than E9 and WB‐aB1 cells. Other growth‐related properties (expression of platelet‐derived growth factor receptor‐β, epidermal growth factor receptor, protein kinase C‐α, protein kinase A regulatory subunit‐Iα, and production of nitric oxide in response to a cocktail of pro‐inflammatory cytokines) were minimally altered or unaffected. Thus, enhancement of connexin expression and GJIC in neoplastic mouse lung and rat liver epithelial cells restored G1 growth control. This was associated with decreased expression of cyclin D1 and increased expression of p27kip‐1, but not with changes in other growth‐related functions. J. Cell. Biochem. 79:347–354, 2000.

Differential polarization of alveolar macrophages and bone marrow-derived monocytes following chemically and pathogen-induced chronic lung inflammation

Alveolar macrophages and BDMCs undergo sequential biochemical changes during the chronic inflammatory response to chemically induced lung carcinogenesis in mice. Herein, we examine two chronic lung inflammation models—repeated exposure to BHT and infection with Mycobacterium tuberculosis—to establish whether similar macrophage phenotype changes occur in non‐neoplastic pulmonary disease. Exposure to BHT or M. tuberculosis results in pulmonary inflammation characterized by an influx of macrophages, followed by systemic effects on the BM and other organs. In both models, pulmonary IFN‐γ and IL‐4 production coincided with altered polarization of alveolar macrophages. Soon after BHT administration or M. tuberculosis infection, IFN‐γ content in BALF increased, and BAL macrophages became classically (M1) polarized, as characterized by increased expression of iNOS. As inflammation progressed in both models, the amount of BALF IFN‐γ content and BAL macrophage iNOS expression decreased, and BALF IL‐4 content and macrophage arginase I expression rose, indicating alternative/M2 polarization. Macrophages present in M. tuberculosis‐induced granulomas remained M1‐polarized, implying that these two pulmonary macrophage populations, alveolar and granuloma‐associated, are exposed to different activating cytokines. BDMCs from BHT‐treated mice displayed polarization profiles similar to alveolar macrophages, but BDMCs in M. tuberculosis‐infected mice did not become polarized. Thus, only alveolar macrophages in these two models of chronic lung disease exhibit a similar progression of polarization changes; polarization of BDMCs was specific to BHT‐induced pulmonary inflammation, and polarization of granuloma macrophages was specific to the M. tuberculosis infection.