Mutagenesis induced by the tumor microenvironment

Genomic instability is a commonly observed feature of tumors. Most investigations addressing the mechanism of tumor progression have focused on the genetic factors that may play a role. Growing evidence now suggests that, in addition to these endogenous factors, the exogenous environment within solid tumors may by itself be mutagenic and constitute a significant source of genetic instability. The tumor microenvironment is characterized by regions of fluctuating hypoxia, low pH, and nutrient deprivation. Each of these microenvironmental factors has been shown to cause severe disturbance in cell metabolism and physiology. Both in vivo and in vitro data demonstrate that exposure of tumor cells to adverse conditions can directly cause mutations, contributing to genetic instability. In this review, we will reexamine the current body of evidence on the role of the tumor microenvironment in inducing mutagenesis and consequent tumor progression.     

Genetic instability induced by the tumor microenvironment

The tumor microenvironment is characterized by regions of fluctuating hypoxia, low pH, and nutrient deprivation. To determine the genetic consequences of growth under these conditions, we used a tumorigenic cell line carrying a recoverable, chromosomally based lambda phage shuttle vector designed to report mutations without the need for genetic selection of mutant cells. The cells were grown in parallel either in culture or as tumors in nude mice. The frequency of mutations arising in cells within the tumors was found to be 5-fold higher than that in otherwise identical cells grown in culture. A distinct pattern of mutation was also seen, with significantly more deletions and transversions in the tumors than in the cell cultures. Furthermore, exposure of the cultured cells to hypoxia produced an elevated mutation frequency and a mutation pattern similar to that seen in the tumors. These results indicate that the conditions within solid tumors are mutagenic and suggest that a fundamental mechanism of tumor progression in vivo is genetic instability induced by the tumor microenvironment.     

Secretion of both IL-2 and IL-4 by tumor cells results in rejection and immunity

Chronic ischemia may cause end stage renal disease, especially in older patients with atherosclerotic renal artery stenosis. Examining the pathology of the ischemic kidney is a fundamental first step toward understanding the mechanisms of this injury. In experimental renal hypoperfusion, there is evidence of a mixture of adaptive responses, tubular and endothelial cell damage and repair events. These processes are reflected in a wide spectrum of morphological changes that include atrophy, focal necrosis, epithelial regeneration, apoptosis, inflammation, interstitial fibrosis, and thrombosis. The most severe damage is seen in the outer medulla, a region with marginal oxygenation even in normal circumstances. In the usual clinical case, the effects of aging, pre-existent hypertension, and the process of atherosclerosis further complicate the pathological picture. Lesions related to these factors include arteriosclerosis, athero-emboli, various types of glomerulosclerosis, and severe tubulointerstitial damage leading to "atubular glomeruli" and regional cortical scarring (nephrosclerosis). In this article, some mechanisms determining the varied and complex pathological findings that may be observed in individual cases are outlined.     

Glial tumor cell proliferation and immune response in the brain

Absence of the maxillary lateral incisor creates an aesthetic problem which can be managed in various ways. The condition requires careful treatment planning and a consideration of the options and outcomes following either space closure or prosthetic replacement. Recent developments in restorative dentistry have warranted a re-evaluation of the approach to this clinical situation. Factors relating both to the patient and to the teeth, including the presenting malocclusion and the effect on the occlusion, are considered. This review considers the possible options: no treatment; orthodontic space closure with canine modification; space maintenance and replacement of the missing tooth with denture, bridge (adhesive and conventional), or implant.     

Effects of estradiol on the expression and production of IGFBP-2 by R3230AC mammary tumor cells

The R3230AC mammary adenocarcinoma of the Fischer rat possesses Type I and Type II IGF receptors. The mRNAs for IGFBP-2, -3, -4, -5 and -6 have been recently identified in this tumor in vivo and in vitro. Using western blotting techniques on tumor tissue homogenates or conditioned media, we demonstrated that IGFBP-2 and, to a lesser extent, IGFBP-3 were expressed, produced, and secreted by the R3230AC tumor cells. Moreover, immunohistochemical assessment of tumor sections with anti-IGFBP-2 demonstrated that signal for IGFBP-2 was localized in the neoplastic glandular epithelium and often in the lumina of the pseudoglandular structures characteristic of this neoplasm. Expression of IGFBP-2 is regulated by the estrogen status of the host. The significant increase occurring in tumors from ovariectomized hosts was completely reversed with hormone repletion. Both mRNA expression and production of IGFBP-2 in vitro were also regulatable by the presence of estradiol-17 beta, with both processes being inhibited by its addition to the cell culture medium. Thus, the response of IGFBP-2 to estrogen showed agreement both in vivo and in vitro, whereas progesterone had no significant effect on these parameters. In the R3230AC tumor, estrogen treatment in vivo decreases tumor growth. Therefore, a relationship could exist between the action of estradiol to inhibit production of IGFBP-2 and the ability of estrogens to regulate tumor growth.     

Antitumor response independent of functional B or T lymphocytes induced by the local and sustained release of interleukin-2 by the tumor cells

Transfection of tumor cells with a vector containing the entire coding sequence of human interleukin-2 (hIL-2) was previously shown to convert the tumorigenic murine fibrosarcoma line CMS-5 into a non-tumorigenic line. The failure of the IL-2-secreting tumor to grow in conventional (immunocompetent) mice was attributed to the activation of CD8+ T cells that exhibited tumor specificity and memory. In order to determine whether or not the IL-2 produced by the tumor may be activating tumor cytotoxic effector cells other than B or T cells we have repeated this study using immunodeficient SCID and SCID-beige mice as syngeneic tumor recipients. In contrast to the rapid growth of the wild-type tumor, the hIL-2-transfected cells (N2A/IL2/CMS5) did not grow, or grew more slowly and regressed, in the mice that lack functional B and T cells. The inhibition of tumor growth associated with the local release of IL-2 was reversed in mice treated with antiasialo-GM1 antibodies specific for natural killer (NK) lineage cells. In contrast to the studies with conventional mice, the IL-2-dependent effector cells in the immunodeficient mice exhibited no evidence of memory. In vitro analysis of spleen cells from tumor-bearing mice revealed the presence of effector cells able to lyse YAC-1 target cells as well as the wild-type CMS-5 and the IL-2-transfected variant tumor lines but unable to lyse P815 cells. The pattern of selective target cell killing and the kinetics of killing were indistinguishable from those observed using tumor necrosis factor alpha (TNF alpha) the mediator associated with natural cytotoxicity cell killing of tumor cells. Histopathology of the IL-2-secreting tumors in SCID mice reveals the presence of infiltrating lymphoid cells and macrophages that were not observed in the CMS-5 tumors. Consistent with the notion that the tumor killing in the SCID mice was mediated by TNF alpha, mice bearing IL-2-secreting tumors had elevated levels of serum TNF alpha and little or no effector cell activity, or TNF alpha was found in tumor-bearing mice treated with anti-asialo-GM1 antibody. The results indicate that the cytokine-induced tumor regression observed in the IL-2-transfected tumors is a more complex phenomenon than previously recognized and one that is mediated by effector cells of the NK cell and/or monocyte/macrophage lineages, in addition to CD8+ T cells.     

Interleukin 1alpha and interleukin 6 promote the in vitro growth of both normal and neoplastic human cervical epithelial cells

We report here on a patient who was diagnosed with follicular carcinoma in 1985, and who was treated with total thyroidectomy. Two years later, when metastasis was found in his neck lesion, lung, pelvis and right femur, the patient received 131I treatment. Six years after receiving 131I treatment, the patient presented with hyperthyroidism. Whole-body scan with 131I revealed functioning metastasis in his right femur and pelvis. There was no hot spot in the neck region, confirming that no thyroid tissue remained. Blood panels revealed an increase in both TSH binding inhibitory immunoglobulin (TBII. 36.2%; normal. -10 approximately 10%) and thyroid stimulating antibody (TSAb, 176%; normal, less than 145%). Treatment with antithyroid drugs, dexamethasone and radioisotope therapy rapidly resolved his hyperthyroidism. Thyrotoxicosis and positive TRAb occurred in the absence of thyroid tissue, and many years after the completion of R1 therapy. The overproduction of thyroid hormone can therefore only be attributed to some mechanism of activity in the metastatic tumor tissue.     

IL-2 adenovector-transduced autologous tumor cells induce antitumor immune responses in patients with neuroblastoma

In many different murine models, the immunogenicity of tumor cells can be increased by transduction with a range of immunostimulatory genes, inducing an immune response that causes regression of pre-existing unmodified tumor cells. To investigate the relevance of these animal models to pediatric malignancy, we used autologous unirradiated tumor cells transduced with an adenovirus-IL-2 to immunize 10 children with advanced neuroblastoma. In a dose-escalation study, we found that this tumor immunogen induced a moderate local inflammatory response consisting predominantly of CD4(+) T lymphocytes, and a systemic response, with a rise in circulating CD25(+) and DR+ CD3(+) T cells. Patients also made a specific antitumor response, manifest by an IgG antitumor antibody and increased cytotoxic T-cell killing of autologous tumor cells. Clinically, five patients had tumor responses after the tumor immunogen alone (one complete tumor response, one partial response, and three with stable disease). Four of these five patients were shown to have coexisting antitumor cytotoxic activity, as opposed to only one of the patients with nonresponsive disease. These results show a promising correlation between preclinical observations and clinical outcome in this disease, and support further exploration of the approach for malignant diseases of children.     

EblacZ tumor dormancy in bone marrow and lymph nodes: active control of proliferating tumor cells by CD8+ immune T cells

A well-defined lacZ gene tagged DBA/2 lymphoma (EblacZ) was used to examine the role of host immune responses in controlling tumor dissemination and persistence, as well as metastasis. In s.c. and intra-ear pinna-inoculated mice, low numbers of EblacZ cells homed to the bone marrow and lymph nodes. The frequency of bone marrow-residing tumor cells did not change with the growth of primary tumor or with multiple inoculations of tumor cells. The bone marrow-residing tumor cells expressed the proliferation-associated Ki67 antigen and expanded upon CD8+ depletion. In contrast, inoculation of nu/nu or severe combined immunodeficiency mice or of immune-suppressed DBA/2 mice led to the rapid outgrowth of EblacZ cells in the bone marrow and their metastasis to other organs. Transfer of bone marrow from EblacZ immunized MHC congenic or syngeneic DBA/2 donors, but not from naive donors, protected s.c.-inoculated DBA/2 mice. Protection was abrogated by in vitro depletion of CD8+ T cells prior to transfer of bone marrow. These experiments show that bone marrow and lymph nodes are privileged sites where potentially lethal tumor cells are controlled in a dormant state by the immune system. Metastasis may be a consequence of the breakdown of this immune control.     

Adenovirus-mediated gene transfer of viral interleukin-10 inhibits the immune response to both alloantigen and adenoviral antigen

Although adenoviral vectors are attractive for gene transfer, their effectiveness is limited by host antiviral immune responses. In this study, we determined if host antiallograft and antiviral immunity could be diminished with an adenoviral vector encoding the immunosuppressive cytokine viral interleukin-10 (vIL-10). AdSV40vIL-10, a vIL-10-expressing adenoviral vector with an SV40 promoter, induced significant prolongation of murine cardiac allograft survival to 32.2 +/- 1.7 days compared to 14.2 +/- 1.0 days for controls (p < 0.01). This effect was specific for vIL-10 encoding vector and could be inhibited by anti-vIL-10 monoclonal antibody (mAb). In vivo administration of adenovirus facilitated the generation of adenovirus-specific cytotoxic T lymphocytes (CTL), whereas treatment with AdSV40vIL-10 prevented CTL priming and generation of virus-specific immunity. AdSV40vIL-10 also induced extended expression of a beta-galactosidase reporter from a co-injected LacZ-encoding adenoviral vector. These results demonstrate that adenovirus-mediated gene transfer and expression of vIL-10 prolong allograft survival and inhibit the immune response to adenoviral antigens, thereby improving the persistence of the vector and extending transgene expression. The efficacy of adenoviral vectors can be improved by incorporating immunosuppressive genes into the vector.     

Induction of cyclooxygenase-2 expression by peroxisome proliferators and non-tetradecanoylphorbol 12,13-myristate-type tumor promoters in immortalized mouse liver cells

Increased expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin synthesis, has been associated with growth regulation and carcinogenesis in several systems. COX-2 is known to be induced by cytokines and the skin tumor promoter 12-tetradecanoylphorbol-13-myristate (TPA). In the present study, we investigated the effects of several non-TPA-type tumor promoters on COX-2 expression in immortalized mouse liver cells. Specifically, we tested peroxisome proliferators (PPs), which are rodent liver tumor promoters that cause gross alterations in cellular lipid metabolism, the rodent liver tumor promoter phenobarbital, and the skin tumor promoters okadaic acid and thapsigargin. The PPs Wy-14643, mono-ethylhexyl phthalate, clofibrate, ciprofibrate ethyl ester, and eicosatetraynoic acid each caused large increases in COX-2 mRNA and protein, with maximal expression seen approximately 10 h after treatment of quiescent cells. COX-2 expression was also induced by thapsigargin, okadaic acid, and calcium ionophore A23187, but not by phenobarbital or the steroid PP dehydroepiandrosterone sulfate. Induction of COX-2 expression generally resulted in increased synthesis of prostaglandin E2 (PGE2). However, the PPs caused little or no increase in PGE2 levels, and they inhibited serum-induced PGE2 synthesis. Unlike non-steroidal anti-inflammatory drugs, the PPs do not directly inhibit cyclooxygenase enzyme activity in vitro. Thus, PPs regulate prostaglandin metabolism via both positive (COX-2 induction) and inhibitory mechanisms. In summary, the strong induction of COX-2 expression by PPs, thapsigargin, and okadaic acid suggests a possible role for COX-2 in the growth regulatory activity of these non-TPA-type tumor promoters.     

Control of the immune response by DHEA and its metabolites

The 17 keto steroid, Dehydroepiandrosterone (5-androsten-3 beta-17-one, DHEA) has been shown to protect mice from a variety of lethal infections. This includes, but is not limited to, infection with viruses (herpesvirus type 2, coxsackievirus B4-CVB4),bacteria (Enterococcus faecalis, Pseudomonas aeruginosa), and a parasite (Cryptosporidium parvum). We have reported that androstenediol (5-androsten-3 beta-17 beta-diol, beta AED), which is derived from DHEA, is at least 100x more effective in up-regulating systemic resistance against CVB4-infection than its precursor. Furthermore, androstenetriol (5-androstene-3 beta-7 beta-17 beta-triol beta AET) which is formed by 7 beta hydroxylation of beta AED, was more effective against CVB4-infection than its precursor beta AED. Neither steroid however has shown any significant direct antiviral effects. The in-vitro influences of DHEA, beta AED, and beta AET on a mitogen-induced mixed splenocyte proliferation assay were determined. The results showed that DHEA suppressed the proliferation of concanavalin A (Con A) or lipopolysaccharide (LPS) activated cultures in a dose dependent manner. beta AED had little influence on the activation response. However, beta AET potentiated the response to both mitogens significantly above control. The regulation of interleukin-2 and interleukin-3 secretion from Con A-activated lymphocytes was analogous to these observations. These functions were suppressed by DHEA, unaffected by beta AED, and potently increased by beta AET. Moreover, the classic immuno-suppressive effects of hydro-cortisone on Con A-induced lymphocyte proliferation, as well as IL-2 and IL-3 production were unaffected by co-cultured with DHEA and only minimally counteracted by beta AED. In contrast, beta AET significantly counteracted the effect of hydrocortisone when co-cultured together. These results show that while in-vivo, DHEA, beta AED, and beta AET each function in a similar manner. In-vitro, their effects are dramatically different from one another with only beta AET potentiating the cellular response by increasing lymphocyte activation and counteracting the immuno-suppressive activity of hydrocortisone.     

Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment

A fundamental obstacle in gene therapy for cancer treatment is the specific delivery of an anticancer gene product to a solid tumor. Although several strategies exist to control gene expression once a vector is directly introduced into a tumor, as yet no systemic delivery system exists that specifically targets solid tumors. Nonpathogenic, obligate anaerobic bacteria of the genus Clostridium have been used experimentally as anticancer agents because of their selective growth in the hypoxic regions of solid tumors after systemic application. In this report we further describe a novel approach to cancer gene therapy in which genetically engineered clostridia are used as tumor-specific vectors for the delivery of antitumor genes. We have introduced into a strain of C. beijerinckii the gene for an E. coli nitroreductase known to activate the nontoxic prodrug CB 1954 to a toxic anticancer drug. Nitroreductase produced by these clostridia enhanced the killing of tumor cells in vitro by CB 1954, by a factor of 22. To demonstrate the specificity of this approach for tumor targeting, we intravenously injected the inactive spore form of C. beijerinckii, which upon transition to a reproductive state will express the E. coli nitroreductase gene. Nitroreductase activity was detectable in 10 of 10 tumors during the first 5 days after intravenous injection of inactive clostridial spores, indicating a rapid transition from spore to reproductive state. Tumors harboring clostridial spores which did not possess the E. coli nitroreductase gene were devoid of nitroreductase activity. Most importantly, E. coli nitroreductase protein was not found in a large survey of normal mouse tissues following intravenous injection of nitroreductase containing clostridia, strongly suggesting that obligate anaerobic bacteria such as clostridia can be utilized as highly specific gene delivery vectors for cancer therapy.     

Transplantation of retinal pigment epithelial cells and immune response in the subretinal space

Theoretical x-ray spectra calculated by four different codes for the same tube parameters are compared by calculating and measuring doses to computed tomography (CT) body and head phantoms. The effect on the 120 kV spectrum, and hence on the calculated dose, of varying the anode angle, tube voltage, and total filtration of the x-ray tube is investigated. Codes used were those of Nowotny and H?fer (XCOMP), Boone, Iles, and Tucker et al. The code based on the work of Tucker et al. produced calculated doses noticeably lower than the other codes and compared best to the measured value. The variation in calculated dose between the Tucker code and the others is of the same order as the variation introduced by uncertainties in total filtration of about 20%, in peak tube voltage of +/- 4 kV, and in change of anode angle from 7 degrees to 13 degrees.     

Antiviral immune responses in mice deficient for both interleukin-2 and interleukin-4

Antiviral immune responses of mice lacking interleukin-2 (IL-2) or IL-4 or both IL-2 and IL-4 (IL-2/4) were compared by using different viruses. Primary cytotoxic T-lymphocyte (CTL) responses against lymphocytic choriomeningitis virus (LCMV) were only moderately reduced in mice lacking IL-2 and were normal in mice lacking IL-4. Mice deficient in both interleukins exhibited variable and more strongly reduced but nevertheless in vivo protective LCMV-specific CTL responses. Similar results were obtained with vaccinia virus. Upon virus-specific restimulation in vitro, spleen cells from IL-2- and IL-2/4-deficient mice failed to generate CTL responses against virus-infected target cells, whereas the response of mice deficient in only IL-4 was comparable to that of control mice. The addition of IL-2 during in vitro restimulation completely restored the responses of both IL-2 and IL-2/4-deficient mice. T-helper-cell-independent immunoglobulin M and T-helper-cell-dependent immunoglobulin G antibody responses against vesicular stomatitis virus glycoprotein were within normal ranges for the various mutant mice. After LCMV infection, specific antibody responses against LCMV nucleoprotein were reduced four- to eightfold. These results show that mice lacking IL-2/4 have an overall tendency to exhibit more severely reduced CTL responses than IL-2- or IL-4-deficient mice. Nevertheless, and surprisingly, in vivo protective immune responses were mounted in the absence of IL-2/4, suggesting that besides a minor contribution from IL-4, other interleukins compensate in vivo for the lack of IL-2 in IL-2-deficient mice.     

Paneth cells and innate immunity in the crypt microenvironment

Paneth cells release granules into the lumen of the crypts of Lieberkuhn in the small intestine where their component proteins participate in mucosal immunity. The granules contain a number of proteins associated with roles in host defense, including lysozyme, secretory phospholipase A2, and alpha-defensins, termed cryptdins. Mouse cryptdins 1-6 and recombinant human Paneth cell alpha-defensin HD-5 are potent antimicrobial agents against certain microorganisms. As defensins, they kill microbes by disruption of the target cell membrane. The peptides are coded by individual, two-exon genes that map to homologous regions of chromosome 8 in mice and humans, and the differential expression of certain mouse cryptdin genes provides markers for studies of crypt ontogeny and epithelial cell differentiation and lineage determination. Neutrophil alpha-defensin peptides exhibit numerous biological activities in addition to antimicrobial function including regulation of cell volume, chemotaxis, mitogenicity, and inhibition of natural killer cell activity. When administered apically, mouse cryptdins 2 and 3 can reversibly stimulate human T-84 intestinal epithelial cells to secrete chloride ion, suggesting that alpha-defensins from Paneth cells also may be multifunctional. Thus, cryptdins and varied Paneth cell secretory products seem to contribute both to innate immunity of the crypt lumen and to defining the apical environment of neighboring cells.