Nancy M. Lee

Id-1 as a molecular target in therapy for breast cancer cell invasion and metastasis

Mammary epithelial cells constitutively expressing Id-1 protein are unable to differentiate, acquire the ability to proliferate, and invade the extracellular matrix. In addition, Id-1 is aberrantly over-expressed in aggressive and metastatic breast cancer cells, as well as in human breast tumor biopsies from infiltrating carcinomas, suggesting Id-1 might be an important regulator of breast cancer progression. We show that human metastatic breast cancer cells become significantly less invasive in vitro and less metastatic in vivo when Id-1 is down-regulated by stable transduction with antisense Id-1. Expression of the matrix metalloproteinase MT1-MMP is decreased in proportion to the decrease in Id-1 protein levels, representing a potential mechanism for the reduction of invasiveness. Further, to more accurately recapitulate the biology of and potential therapeutic approaches to tumor metastasis, we targeted Id-1 expression systemically in tumor-bearing mice by using a nonviral approach. We demonstrate significant reduction of both Id-1 and MT1-MMP expressions as well as the metastatic spread of 4T1 breast cancer cells in syngeneic BALB/c mice. In conclusion, our studies have identified Id-1 as a critical regulator of breast cancer progression and suggest the feasibility of developing novel therapeutic approaches to target Id-1 expression to reduce breast cancer metastasis in humans.

Role of zinc in ALS

The causes of amyotrophic lateral sclerosis (ALS) are poorly understood. A small proportion, about 2%, is associated with a mutation in the superoxide dismutase (SOD1) gene, and mice expressing this mutant gene exhibit a progressive, ALS‐like neurodegenerative disease. Studies of these animals, as well as of human post mortem tissue, reveal the presence of multiple pathological processes, including oxidative stress, glutamate excitotoxicity, neuroinflammation, mitochondrial degeneration, alterations in neurofilaments and neurotubules, mitochondrial damage, aggregation of proteins, abnormalities in growth factors, and apoptosis. We propose that alterations in the disposition of zinc ions may be important in the initiation and development of ALS. SOD1 binds zinc, and many of the mutant forms of this enzyme associated with ALS show altered zinc binding. Alterations in the expression of metallothioneins (MTs), which regulate cellular levels of zinc, have been reported in mutant SOD1 mice, and deletion of MTs in these animals accelerates disease progression. Zinc plays a key role in all the pathological processes associated with ALS. Our zinc hypothesis also may help explain evidence for environmental factors in some cases of ALS, such as in the Chamorro tribe in Guam and in the Gulf War.

Opioid receptor interactions: Local and nonlocal, symmetric and asymmetric, physical and functional

The pharmacological effects of opioid drugs and endogenous opioid peptides are mediated by several kinds of receptors, the major ones being mu, delta and kappa. Though classically it has been thought that a particular effect mediated by a drug or other ligand results from its interaction with a single type of receptor, accumulating evidence demonstrates that interactions between receptors play a major role in opioid actions. These interactions may be either local, involving receptors within the same tissue, or nonlocal, between receptors located in different tissues. Nonlocal interactions always involve intercellular mechanisms, whereas local interactions may involve either intercellular or intracellular interactions, the latter including physical association of receptors. Both local and nonlocal interactions, moreover, may be either symmetric, with ligand interaction at one receptor affecting interaction at the other, or asymmetric; and either potentiating or inhibitory. In this article we discuss major examples of these kinds of interactions, and their role in the acute and chronic effects of opioids. Knowledge of these interactions may have important implications for the design of opioids with more specific actions, and for eliminating the addictive liability of these drugs.

Altered gene expression in normal colonic mucosa of individuals with polyps of the colon.

PURPOSEExpression levels of many genes are altered in colon cancer, relative to normal colonic mucosa. We recently reported that such differences also exist between grossly normal colonic mucosa of individuals with and without colon cancer, and between individuals with and without a family history of colon cancer. Here we report a study of individuals with no cancer but with polyps in the transverse, ascending/descending, or rectosigmoid colon.METHODSBiopsies of grossly normal-appearing colonic mucosa from the rectosigmoid colon were taken from individuals with polyps, with or without personal/family history of colon cancer, and gene expression profiles compared with those from biopsies of control patients, with no polyps or known personal/family history. A global expression analysis was conducted of the same 15 genes used in our previous studies.RESULTSWe found significant differences in gene expression in normal-appearing rectosigmoid colonic mucosa between individuals with polyps and controls, regardless of whether personal or family history of cancer was present. CONCLUSIONSAlterations in gene expression patterns in morphologically normal-appearing colonic mucosa are associated with the presence of adenomatous polyps. Prospective studies will be required to determine whether these alterations in gene expression can be used to predict risk of developing colon cancer.

Temporal gene expression patterns in G93A/SOD1 mouse

Amyotrophic lateral sclerosis (ALS) is a generally fatal degenerative disorder of motor neurons that has no known cure. Many pathological processes have been implicated. However, the early, initiating events in the disease are poorly understood. We performed multivariate analyses of gene expression of 21 selected genes from categories including glutamate neurotoxicity, oxidative stress, neuroinflammation, aberrant metal ion regulation, apoptosis, and abnormal microglial function on G93A SOD1 mice. These animals develop symptoms of motor neuron dysfunction at about 12 weeks of age, and die at age 18 to 20 weeks. We analyzed animals at both presymptomatic and symptomatic stages. Differential regulation of several genes involved in neuroinflammation, including TNF‐α, IL‐ RA, CD86, CD200R and Groα, was observed in presymptomatic mice, aged 6–9 weeks, while expression of genes representative of other processes was not altered until the animals reached symptomatic stages. Analysis of expression of inflammatory genes and microglia related genes together also revealed a highly significant change in mutant mice relative to wildtype at 6–9 weeks. These changes were due to the presence of the mutant gene, and not simply to overexpression of a SOD1 gene. These findings are discussed in relation to possible roles of microglia function in the development of ALS.

Non-opioid dynorphin binding site on secretory vesicles of a pituitary-derived cell line

Accumulating evidence indicates that the endogenous opioid peptides dynorphinA-(1-17) and dynorphinA-(1-13) interact not only with opioid but also with yet poorly characterized non-opioid receptors. The latter have been implicated in a number of the effects of dynorphins including induction of ACTH release in sheep and in AtT 20 cells, a pituitary-derived mouse cell line. AtT 20 cells do not express opioid receptors and therefore are particularly suitable for search of non-opioid dynorphin receptors. We report here that 3H-dynorphinA-(1-13)-NH2 associates specifically with AtT 20 cells, apparently through an uptake process and a binding site. Within the cell, it binds preferentially to fractions containing secretory vesicles, with a Kd of about 100 nM. DynorphinA-(1-17), and several non-opioid fragments of dynorphin, including A-(2-17), A-(2-16) and A-(2-13), compete with 3H-dynorphinA-(1-13)-NH2 for that site with IC50s ranging from 200 nM to 2 microM. ACTH(1-39) also competes with 3H-dynorphinA-(1-13)-NH2 for the site with an IC50 of about 300 nM. DynorphinA-(2-17) at microM concentrations stimulates release of ACTH from the isolated vesicles. The results indicate the presence of a non-opioid dynorphin binding site on the secretory vesicle fractions of AtT20 cells that might be involved in ACTH release. The ability of ACTH itself to compete for the binding sites associated with the vesicles suggest that those sites may be involved in an autocrine loop.

Different real-time PCR systems yield different gene expression values

Most polymerase chain reaction (PCR) systems employ pre-determined settings and proprietary master mixes that differ from one system to another. It is not known whether these differences may affect gene expression values. We compared two major real-time PCR technologies, from Life Technologies (formerly Applied Biosystems; ABI7500) and Roche Applied Science (LC480), using their default settings, proprietary reagents and other potential variables such as ramp rates and magnesium concentrations. We analyzed four genes (IL-8, COX2, ID-1 and CXCR2) in a human breast cancer cell line and found that two of them, though readily detected by ABI, were not detected using the Roche system. By altering some of the parameters and reagents used in the Roche protocol, we were able to detect expression of these two genes, but the level remained far below that detected by ABI, particularly for ID-1. When we tested three additional ID-1 primer pairs, two of these primer pairs yielded higher expression values in the LC system, yet still significantly lower than the values obtained in ABI. These results suggest critical differences in these two PCR systems, which could result in significant discrepancies in results reported by different laboratories.

Tolerance develops in spinal cord, but not in brain with chronic [Dmt1]DALDA treatment

Previously, we reported that H‐2′,6′‐dimethyltyrosine [Dmt1]‐D‐Arg‐Phe‐Lys‐NH2 (DALDA), an analogue of the naturally occurring opioid peptide dermorphin, is a highly potent and selective mu receptor agonist with low cross‐tolerance to morphine. In the present study, we investigated the effect of treating mice chronically with [Dmt1]DALDA. The AD50 of [Dmt1]DALDA (s.c.) increased eight‐fold in animals given this drug chronically; in contrast, the AD50 increased two‐fold in mice chronically treated with morphine. The AD50 of morphine (s.c.) in these [Dmt1]DALDA‐treated animals was increased more than 120 times, while that of the more selective μ agonist [D‐Ala2‐MePhe4‐Gly‐ol5]enkephalin (DAMGO) given intrathecally was increased more than 240 times. However, the AD50 of DAMGO given intracerebroventricularly was essentially the same in animals treated chronically with [Dmt1]DALDA as in naïve animals. The dose of naloxone required to precipitate withdrawal in [Dmt1]DALDA‐treated animals was 20 times lower than that in morphine‐tolerant animals. Using real‐time quantitative PCR, we found that expression of the μ opioid receptor, δ opioid receptor, preproenkephalin and preprodynorphin genes was upregulated in the brain by [Dmt1]DALDA treatment. No significant changes in expression of opioid receptor or opioid peptide genes were detected in the spinal cord of [Dmt1]DALDA‐treated mice, nor in the brain or spinal cord of morphine‐treated mice. We conclude that a high degree of tolerance to [Dmt1]DALDA develops in the spinal cord but not brain, and cannot be accounted for by changes in expression of opioid receptors or opioid peptides in these tissues.

Opioid receptor polymorphismsand opioid abuse

The sequencing of the human genome is only the first step. The next step is to determine the function of these genes and in particular, how alterations in specific genes lead to major human disorders. Many laboratories are now focusing on identifying and characterizing single nucleotide polymorphisms (SNPs), to determine which correlate in frequency with certain population groups who may be particularly susceptible to certain diseases. The mu opioid receptor (MOR), which mediates the clinically important analgesic effects of drugs like morphine as well as the euphoria sought by heroin abusers, exhibits several dozen polymorphisms. Several of these are associated with altered receptor function and individuals at risk for drug abuse.

The helix-loop-helix Id-1 inhibits PSA expression in prostate cancer cells

The inhibitor of basic helix‐loop‐helix transcription factors, Id‐1, is an important gene whose expression increases during prostate cancer progression and that upregulates proliferation, migration and invasion. We used microarray analysis to identify the downstream genes whose transcriptional expression is modulated by Id‐1 protein. We compared gene expression in control LNCaP cells and Id‐1‐transduced LNCaP cells, which become significantly more aggressive after Id‐1 overexpression, thus mimicking the high levels of Id‐1 detected in metastatic cell lines. We used the Affy HTA U133A Expression Arrays with 45,000 probe sets representing more than 39,000 transcripts. We found that one of the most significantly downregulated genes on Id‐1 expression was kallikrein 3 [also called prostate specific antigen (PSA)], the most commonly used biomarker of prostate cancer. Here, we show that the reduction in PSA mRNA and protein expression associated with high‐grade prostate cancers, which generally express high levels of Id‐1, could be the consequence of Id‐1 overexpression.