A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation.

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity.

A Gene Expression Signature Associated with Overall Survival in Patients with Hepatocellular Carcinoma Suggests a New Treatment Strategy.

Despite improvements in the management of liver cancer, the survival rate for patients with hepatocellular carcinoma (HCC) remains dismal. The survival benefit of systemic chemotherapy for the treatment of liver cancer is only marginal. Although the reasons for treatment failure are multifactorial, intrinsic resistance to chemotherapy plays a primary role. Here, we analyzed the expression of 377 multidrug resistance (MDR)-associated genes in two independent cohorts of patients with advanced HCC, with the aim of finding ways to improve survival in this poor-prognosis cancer. Taqman-based quantitative polymerase chain reaction revealed a 45-gene signature that predicts overall survival (OS) in patients with HCC. Using the Connectivity Map Tool, we were able to identify drugs that converted the gene expression profiles of HCC cell lines from ones matching patients with poor OS to profiles associated with good OS. We found three compounds that convert the gene expression profiles of three HCC cell lines to gene expression profiles associated with good OS. These compounds increase histone acetylation, which correlates with the synergistic sensitization of those MDR tumor cells to conventional chemotherapeutic agents, including cisplatin, sorafenib, and 5-fluorouracil. Our results indicate that it is possible to modulate gene expression profiles in HCC cell lines to those associated with better outcome. This approach also increases sensitization of HCC cells toward conventional chemotherapeutic agents. This work suggests new treatment strategies for a disease for which few therapeutic options exist.

Dragging ras back in the ring.

Ras proteins play a major role in human cancers but have not yielded to therapeutic attack. Ras-driven cancers are among the most difficult to treat and often excluded from therapies. The Ras proteins have been termed "undruggable," based on failures from an era in which understanding of signaling transduction, feedback loops, redundancy, tumor heterogeneity, and Ras' oncogenic role was poor. Structures of Ras oncoproteins bound to their effectors or regulators are unsolved, and it is unknown precisely how Ras proteins activate their downstream targets. These knowledge gaps have impaired development of therapeutic strategies. A better understanding of Ras biology and biochemistry, coupled with new ways of targeting undruggable proteins, is likely to lead to new ways of defeating Ras-driven cancers.

Intra-individual variability over time in serum cytokine levels among participants in the prostate, lung, colorectal, and ovarian cancer screening Trial.

BACKGROUND: Serum measurements of cytokines, mediators of various B and T cell activities, are important markers of inflammation and immune dysregulation. We assessed the reproducibility of serum cytokine measurements over a five-year period among participants in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). METHODS: Levels of 13 cytokines [interleukin (IL) 1ß, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, interferon-gamma (IFNγ), granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNFα)] in stored sera from three collections (study baseline,+1 year, and+5 years) among 28 randomly selected PLCO participants were measured using a high-sensitivity Luminex xMap-based multiplex panel. Within- and between-subject components of variance were estimated from random effects models and were used to calculate the coefficient of variation (CV) and intraclass correlation coefficient (ICC) for analytes with <30% of samples below the limit of detection (LOD). Spearman correlation coefficients between measurements of the same analyte over time and between analytes were also calculated. RESULTS: Among the six cytokines with <30% of samples below the LOD, we observed excellent reproducibility for IL-6, IL-7, IL-13, and TNFα (ICC≥0.73), and fair to good reproducibility for IL-8 (ICC=0.55) and IL-10 (ICC=0.60). Spearman correlation coefficients comparing paired measurements of each cytokine at baseline and at +5 years were high (ρ≥0.74) with the exception of IL-10 (ρ=0.44). CONCLUSIONS: These results suggest that measurements of most of the cytokines evaluated in this study were highly reproducible over five-year periods.

Purify First: rapid expression and purification of proteins from XMRV.

Purifying proteins from recombinant sources is often difficult, time-consuming, and costly. We have recently instituted a series of improvements in our protein purification pipeline that allows much more accurate choice of expression host and conditions and purification protocols. The key elements are parallel cloning, small scale parallel expression and lysate preparation, and small scale parallel protein purification. Compared to analyzing expression data only, results from multiple small scale protein purifications predict success at scale-up with greatly improved reliability. Using these new procedures we purified eight of nine proteins from xenotropic murine leukemia virus-related virus (XMRV) on the first attempt at large scale.

Kaposi sarcoma (KS)-associated herpesvirus microRNA sequence analysis and KS risk in a European AIDS-KS case control study.

BACKGROUND: We recently identified polymorphisms in Kaposi sarcoma-associated herpesvirus (KSHV)-encoded microRNA (miRNA) sequences from clinical subjects. Here, we examine whether any of these may contribute to KS risk in a European AIDS-KS case-control study. METHODS: KSHV load in peripheral blood was determined by real-time quantitative polymerase chain reaction. Samples that had detectable viral loads were used to amplify the 2.8-kb miRNA encoding region plus a 646-bp fragment of the K12/T0.7 gene. Additionally, we characterized an 840-bp fragment of the K1 gene to determine KSHV subtypes. RESULTS: KSHV DNA was detected in peripheral blood mononuclear cells of 49.6% of case patients and 6.8% of controls, and viral loads tended to be higher in case patients. Sequences from the miRNA-encoding regions were conserved overall, but distinct polymorphisms were detected, some of which occurred in primary miRNAs, pre-miRNAs, or mature miRNAs. CONCLUSIONS: Patients with KS were more likely to have detectable viral loads than were controls without disease. Despite high conservation in KSHV miRNA-encoded sequences, polymorphisms were observed, including some that have been reported elsewhere. Some polymorphisms could affect mature miRNA processing and appear to be associated with KS risk.

Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a debilitating disease of unknown etiology that is estimated to affect 17 million people worldwide. Studying peripheral blood mononuclear cells (PBMCs) from CFS patients, we identified DNA from a human gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), in 68 of 101 patients (67%) as compared to 8 of 218 (3.7%) healthy controls. Cell culture experiments revealed that patient-derived XMRV is infectious and that both cell-associated and cell-free transmission of the virus are possible. Secondary viral infections were established in uninfected primary lymphocytes and indicator cell lines after their exposure to activated PBMCs, B cells, T cells, or plasma derived from CFS patients. These findings raise the possibility that XMRV may be a contributing factor in the pathogenesis of CFS.

Reactivation of Kaposi's sarcoma-associated herpesvirus by natural products from Kaposi's sarcoma endemic regions.

Kaposi's sarcoma (KS) and its causative agent, Kaposi's sarcoma associated herpesvirus (KSHV/HHV-8), a gamma2 herpesvirus, have distinctive geographical distributions that are largely unexplained. We propose the "oncoweed" hypothesis to explain these differences, namely that environmental cofactors present in KS endemic regions cause frequent reactivation of KSHV in infected subjects, leading to increased viral shedding and transmission leading to increased prevalence of KSHV infection as well as high viral load levels and antibody titers. Reactivation also plays a role in the pathogenesis of KSHV-associated malignancies. To test this hypothesis, we employed an in vitro KSHV reactivation assay that measured increases in KSHV viral load in KSHV infected primary effusion lymphoma (PEL) cells and screened aqueous natural product extracts from KS endemic regions. Of 4,842 extracts from 38 countries, 184 (5%) caused KSHV reactivation. Extracts that caused reactivation came from a wide variety of plant families, and extracts from Africa, where KSHV is highly prevalent, caused the greatest level of reactivation. Time course experiments were performed using 28 extracts that caused the highest levels of reactivation. The specificity of the effects on viral replication was examined using transcriptional profiling of all viral mRNAs. The array data indicated that the natural extracts caused an ordered cascade of lytic replication similar to that seen after induction with synthetic activators. These in vitro data provide support for the "oncoweed" hypothesis by demonstrating basic biological plausibility.

Hepatitis C virus genotype 4 in Ugandan children and their mothers.

In Kampala, Uganda, in 2001, hepatitis C virus antibodies were found in 27 (4%) of 603 children and in 62 (12%) of 525 of their mothers. However, only approximately 10% of positive results were confirmed by reverse transcription-PCR, which suggests frequent false-positive results or viral clearance. All sequenced types were genotype 4.

Molecular evidence for mother-to-child transmission of Kaposi sarcoma-associated herpesvirus in Uganda and K1 gene evolution within the host.

BACKGROUND: Epidemiological studies of Kaposi sarcoma (KS)-related herpesvirus (KSHV) indicate that having a KSHV-seropositive mother is a risk factor for KSHV infection in children. METHODS: We determined the KSHV K1 sequences in concordantly polymerase chain reaction-positive Ugandan mother-child pairs, to ascertain whether they shared the same viral strain. We also examined sequences amplified from saliva and buffy coat samples from the same subjects, to investigate potential intrasubject sequence differences. RESULTS: We obtained K1 sequences from 6 of 10 mother-child pairs. In 1 pair, the subtypes differed between mother and child. The mother and child in 2 other pairs shared the same subtype, but the sequences differed. The mother and child in 2 pairs shared KSHV strains with exact (100%) nucleotide homology. The last pair showed evidence of viral strain concordance between mother and child but also showed evidence of evolution of the viral sequence within the child. Of 26 study subjects, 19 showed no evidence of intrasubject K1 sequence variability, but, in 7 subjects, all of whom were children, amino acid variation of 1%-4% was observed. CONCLUSIONS: Our findings are consistent with KSHV transmission from maternal and nonmaternal sources in KS-endemic regions. Our results also provide evidence for ongoing evolution of the K1 gene in KSHV-infected children.

Genotypic characterization of Kaposi's sarcoma-associated herpesvirus in asymptomatic infected subjects from isolated populations.

Molecular epidemiological studies of Kaposi's sarcoma-associated herpesvirus (KSHV) have concentrated on characterization of viral strains in tumour biopsy samples from Kaposi's sarcoma (KS) patients, mostly obtained in the United States and Europe. Tumour biopsies are a convenient source of viral DNA, as they have a high viral load compared to peripheral blood. However, sequences obtained from biopsies may not be representative of viral strains in asymptomatic subjects and information on ethnicity is often not available. Here, a population-based approach has been used to study the molecular and seroepidemiology of KSHV in isolated populations in Ecuador and Botswana. Amerindians in Ecuador had a variable prevalence of KSHV and all strains characterized were of subtype E, based on K1 sequencing. All Amerindian strains had predominant (P)-type K15 alleles and had sequences in both T0.7 and ORF 75 that appeared to be characteristic of these strains. The prevalence of KSHV in two ethnic groups in Botswana was extremely high. K1 sequences from both Bantu and San subjects were mostly of subtypes B and A5, which are typical of African KSHV strains, but the sequence from one San subject did not cluster with any known subtype. Considerable heterogeneity was seen in the T0.7 and ORF 75 genes in the San subjects and one had a minor (M)-type K15 allele. The heterogeneity of the KSHV strains found in these subjects from Botswana contrasts with the homogeneity of KSHV strains in Amerindians, reflecting differences in the evolutionary history of these populations.