Kathleen L. McGuire

IL-2 activation of a PI3K-dependent STAT3 serine phosphorylation pathway in primary human T cells.

Interleukin-2 (IL-2) is the major growth factor of activated T lymphocytes. By inducing cell cycle progression and protection from apoptosis in these cells, IL-2 is involved in the successful execution of an immune response. Upon binding its receptor, IL-2 activates a variety of signal transduction pathways, including the Ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and Janus kinase (JAK)/STAT cascades. In addition, activation of phosphatidylinositol 3-kinase (PI3K) and several of its downstream targets has also been shown. However, the coupling of STAT3 serine phosphorylation to PI3K in response to IL-2 has yet to be shown in either T cell lines or primary human T cells. This report shows that the PI3K inhibitors LY294002 and wortmannin block activation of MEK and ERK by IL-2 in primary human T cells. Moreover, these inhibitors significantly reduce IL-2-triggered STAT3 serine phosphorylation without affecting STAT5 serine phosphorylation. Analysis of the effects of these inhibitors on cell cycle progression and apoptosis strongly suggests that PI3K-mediated events, which includes STAT3 activation, are involved in IL-2-mediated cell proliferation but not cell survival. Finally, results presented illustrate that in primary human T cells, activation of Akt is insufficient for IL-2-induced anti-apoptosis. Thus, these results demonstrate that IL-2 stimulates PI3K-dependent events that correlate with cell cycle progression, but not anti-apoptosis, in activated primary human T cells.

Repression of IL-2 Promoter Activity by the Novel Basic Leucine Zipper p21SNFT Protein

IL-2 is the major autocrine and paracrine growth factor produced by T cells upon T cell stimulation. The inducible expression of IL-2 is highly regulated by multiple transcription factors, particularly AP-1, which coordinately activate the promoter. Described here is the ability of the novel basic leucine zipper protein p21SNFT to repress AP-1 activity and IL-2 transcription. A detailed analysis of the repression by p21SNFT repression on the IL-2 promoter distal NF-AT/AP-1 site demonstrates that it can bind DNA with NF-AT and Jun, strongly suggesting that it represses NF-AT/AP-1 activity by competing with Fos proteins for Jun dimerization. The importance of this repression is that p21SNFT inhibits the trans-activation potential of protein complexes that contain Jun, thereby demonstrating an additional level of control for the highly regulated, ubiquitous AP-1 transcription factor and the IL-2 gene.

Comprehensive Study of Sansalvamide A Derivatives and their Structure–Activity Relationships against Drug-Resistant Colon Cancer Cell Lines

We report an extensive structure-activity relationship (SAR) of 62 compounds active against two drug-resistant colon cancer cell lines. Our comprehensive evaluation of two generations of compounds utilizes SAR, NMR, and molecular modeling to evaluate the key 3D features of potent compounds. Of the seven most potent compounds reported here, five are second-generation, emphasizing our ability to incorporate potent features found in the first generation and utilize their structures to design potency into the second generation. These analogs share no structural homology to current colon cancer drugs, are cytotoxic at levels on par with existing drugs treating other cancers, and demonstrate selectivity for drug-resistant colon cancer cell lines over noncancerous cell lines. Thus, we have established sansalvamide A as an excellent lead for treating multiple drug-resistant colon cancers.

Influence of race on microsatellite instability and CD8+ T cell infiltration in colon cancer

African American patients with colorectal cancer show higher mortality than their Caucasian counterparts. Biology might play a partial role, and prior studies suggest a higher prevalence for microsatellite instability (MSI) among cancers from African Americans, albeit patients with MSI cancers have improved survival over patients with non-MSI cancers, counter to the outcome observed for African American patients. CD8+ T cell infiltration of colon cancer is postively correlated with MSI tumors, and is also related to improved outcome. Here, we utilized a 503-person, population-based colon cancer cohort comprising 45% African Americans to determine, under blinded conditions from all epidemiological data, the prevalence of MSI and associated CD8+ T cell infiltration within the cancers. Among Caucasian cancers, 14% were MSI, whereas African American cancers demonstrated 7% MSI (P = 0.009). Clinically, MSI cancers between races were similar; among microsatellite stable cancers, African American patients were younger, female, and with proximal cancers. CD8+ T cells were higher in MSI cancers (88.0 vs 30.4/hpf, P<0.0001), but was not different between races. Utilizing this population-based cohort, African American cancers show half the MSI prevalence of Caucasians without change in CD8+ T cell infiltration which may contribute towards their higher mortality from colon cancer.

IL-2- and STAT5-regulated cytokine gene expression in cells expressing the Tax protein of HTLV-1

Interleukin-2 (IL-2) mediates cell cycle progression and antiapoptosis in human T cells via several signal transduction pathways. The Tax protein of the human T-cell leukemia virus type I (HTLV-1) deregulates cell growth and alters the role of IL-2 in infected cells. However, Tax-immortalized cells stay dependent on IL-2, suggesting that events besides HTLV-1 gene expression are required for leukemia to develop. Here, IL-2-dependent and -independent events were analysed in a human T cell line immortalized by Tax. These studies show that, of the signaling pathways evaluated, only STAT5 remains dependent. Microarray analyses revealed several genes, including il-5, il-9 and il-13, are uniquely upregulated by IL-2 in the presence of Tax. Bioinformatics and supporting molecular biology show that some of these genes are STAT5 targets, explaining their IL-2 upregulation. These results suggest that IL-2 and viral proteins work together to induce gene expression, promoting the hypothesis that deregulation via the constitutive activation of STAT5 may lead to the IL-2-independent phenotype of HTLV-1-transformed cells.

Expression differences between African American and Caucasian prostate cancer tissue reveals that stroma is the site of aggressive changes

In prostate cancer, race/ethnicity is the highest risk factor after adjusting for age. African Americans have more aggressive tumors at every clinical stage of the disease, resulting in poorer prognosis and increased mortality. A major barrier to identifying crucial gene activity differences is heterogeneity, including tissue composition variation intrinsic to the histology of prostate cancer. We hypothesized that differences in gene expression in specific tissue types would reveal mechanisms involved in the racial disparities of prostate cancer. We examined 17 pairs of arrays for AAs and Caucasians that were formed by closely matching the samples based on the known tissue type composition of the tumors. Using pair‐wise t‐test we found significantly altered gene expression between AAs and CAs. Independently, we performed multiple linear regression analyses to associate gene expression with race considering variation in percent tumor and stroma tissue. The majority of differentially expressed genes were associated with tumor‐adjacent stroma rather than tumor tissue. Extracellular matrix, integrin family and signaling mediators of the epithelial‐to‐mesenchymal transition (EMT) pathways were all downregulated in stroma of AAs. Using MetaCore (GeneGo) analysis, we observed that 35% of significant (p < 10−3) pathways identified EMT and 25% identified immune response pathways especially for interleukins‐2, ‐4, ‐5, ‐6, ‐7, ‐10, ‐13, ‐15 and ‐22 as the major changes. Our studies reveal that altered immune and EMT processes in tumor‐adjacent stroma may be responsible for the aggressive nature of prostate cancer in AAs.

Correlation of transcriptional repression by p21SNFT with changes in DNA.NF-AT complex interactions

p21SNFT (21-kDa small nuclear factor isolated from T cells) is a novel human protein of the basic leucine zipper family. The overexpression of p21SNFTleads to the significant and specific repression of transcription from the interleukin-2 promoter as well as from several essential activator protein 1 (AP-1)-driven composite promoter elements. One example is the distal nuclear factor of activated T cells (NF-AT)/AP-1 element where the AP-1 (Fos/Jun) basic leucine zipper heterodimer interacts with members of the NF-AT family. p21SNFT has been shown to replace Fos in dimerization with Jun on a consensus AP-1 binding site (12-O-tetradecanolyphorbol-13-acetate response element (TRE)) and to interact with Jun and NF-AT at the distal NF-AT/AP-1 enhancer element. A detailed biochemical analysis presented here compares interactions involving p21SNFT with those involving Fos. The results demonstrate that a p21SNFT/Jun dimer binds a TRE similarly to AP-1 and like AP-1 binds cooperatively with NF-AT at the NF-AT/AP-1 composite element. However, Fos interacts significantly more efficiently than p21SNFT with Jun and NF-AT, and the replacement of Fos by p21SNFT in the trimolecular complex drastically alters protein-DNA contacts. The data suggest that p21SNFT may repress transcriptional activity by inducing a unique conformation in the transcription factor complex.

Immune responses elicited by bacterial minicells capable of simultaneous DNA and protein antigen delivery

Abstract Recent events surrounding emerging infectious diseases, bioterrorism and increasing multidrug antibiotic resistance in bacteria have drastically increased current needs for effective vaccines. Many years of study have shown that live, attenuated pathogens are often more effective at delivering heterologous protein or DNA to induce protective immune responses. However, these vaccine carriers have inherent safety concerns that have limited their development and their use in many patient populations. Studies using nonliving delivery mechanisms have shown that providing both protein antigen and DNA encoding the antigen to an individual induces an improved, more protective immune response but rarely, if ever, are both delivered simultaneously. Here, non-replicating bacterial minicells derived from a commensal E. coli strain are shown to effectively induce antigen-specific immune responses after simultaneous protein and DNA delivery. These data demonstrate the potential use of achromosomal bacterial minicells as a vaccine carrier.

Macrophages influence Salmonella host-specificity in vivo

The generalist Salmonella enterica serovar Typhimurium causes disease in many animal species, but the closely related host-specific serovar Typhi only causes disease in humans. Typhi and Typhimurium share major virulence loci; hence it is not known exactly why Typhi does not cause disease in mice. We tested the hypothesis that macrophages contribute to Salmonella host-specificity in mice. No significant difference in survival of the two serovars was observed in vitro in mouse macrophage cell lines and primary murine peritoneal and bone marrow-derived macrophages after 24h. In contrast, differential survival was observed following infection in vivo. When BALB/c mice were infected intraperitoneally (i.p.), both Typhi and Typhimurium induced neutrophil influx into the peritoneum and macrophages were the major cell type containing internalized bacteria at 0.5 and 4h post-infection for both serovars. The number of Typhimurium in macrophages remained high at 4h post-infection, but the number of Typhi in macrophages decreased substantially within 4h after i.p. infection. These results indicate that macrophages are able to distinguish Typhi from Typhimurium when infected in vivo but no significant differences were observed after 24h in vitro, suggesting that the differential killing of the two serovars by macrophages requires additional factors within the host.

Transcriptional repression of MMP-1 by p21SNFT and reduced in vitro invasiveness of hepatocarcinoma cells

p21SNFT (21 kDa small nuclear factor isolated from T cells) is a human basic leucine zipper transcription factor that can repress AP-1-mediated transcription. We show here that overexpression of p21SNFT in HepG2 cells leads to repression of matrix metalloproteinase-1 by 70–80%. p21SNFT interacted with Jun at the matrix metalloproteinase-1 promoter −88 Ets/AP-1 enhancer element, where Jun is known to activate transcription via interaction with Fos and Ets proteins. When p21SNFT/Jun dimers bound the element in the presence of Ets, DNA was protected differently than when Fos was paired with Jun. The data suggest a difference in overall conformation between p21SNFT-containing and Fos-containing complexes that may be involved in the repression of matrix metalloproteinase-1 by p21SNFT. Overexpression of p21SNFT led to a reduction in invasiveness of HepG2 cells through type I collagen and reconstituted basement membrane, an effect similar to that obtained via direct immunodepletion of matrix metalloproteinase-1. The results indicate that the mechanism of repression of matrix metalloproteinase-1 by p21SNFT may be exploited in inhibiting pathological matrix remodeling during cancer progression in vivo.