Identification of mutant K-Ras-dependent phenotypes using a panel of isogenic cell lines.

To assess the consequences of endogenous mutant K-Ras, we analyzed the signaling and biological properties of a small panel of isogenic cell lines. These include the cancer cell lines DLD1, HCT116, and Hec1A, in which either the WT or mutant K-ras allele has been disrupted, and SW48 colorectal cancer cells and human mammary epithelial cells in which a single copy of mutant K-ras was introduced at its endogenous genomic locus. We find that single copy mutant K-Ras causes surprisingly modest activation of downstream signaling to ERK and Akt. In contrast, a negative feedback signaling loop to EGFR and N-Ras occurs in some, but not all, of these cell lines. Mutant K-Ras also had relatively minor effects on cell proliferation and cell migration but more dramatic effects on cell transformation as assessed by growth in soft agar. Surprisingly, knock-out of the wild type K-ras allele consistently increased growth in soft agar, suggesting tumor-suppressive properties of this gene under these conditions. Finally, we examined the effects of single copy mutant K-Ras on global gene expression. Although transcriptional programs triggered by mutant K-Ras were generally quite distinct in the different cell lines, there was a small number of genes that were consistently overexpressed, and these could be used to monitor K-Ras inhibition in a panel of human tumor cell lines. We conclude that there are conserved components of mutant K-Ras signaling and phenotypes but that many depend on cell context and environmental cues.

A requirement for wild-type Ras isoforms in mutant KRas-driven signalling and transformation.

The mutant forms of KRas, NRas and HRas drive the initiation and progression of a number of human cancers, but less is known about the role of WT (wild-type) Ras alleles and isoforms in cancer. We used zinc-finger nucleases targeting HRas and NRas to modify both alleles of these genes in the mutant KRas-driven Hec1A endometrial cancer cell line, which normally expresses WT copies of these genes. The disruption of either WT isoform of Ras compromised growth-factor-dependent signalling through the ERK (extracellular-signal-regulated kinase) pathway. In addition, the disruption of HRas hindered the activation of Akt and subsequent downstream signalling. This was associated with decreased proliferation, increased apoptosis and decreased anchorage-independent growth in the HRas-disrupted cells. However, xenograft tumour growth was not significantly affected by the disruption of either NRas or HRas. As expected, deleting the mutant allele of KRas abolished tumour growth, whereas deletion of the remaining WT copy of KRas increased the tumorigenic properties of these cells; deleting a single copy of either HRas or NRas did not mimic this effect. The present study demonstrates that the WT copies of HRas, NRas and KRas play unique roles in the context of mutant KRas-driven tumours.

Age-based normative data for a computerized dynamic posturography system that uses a virtual visual surround environment.

BACKGROUND: Computerized dynamic posturography (CDP) is the gold standard to differentiate between sensory, motor, and central adaptive impairments to postural control. OBJECTIVE: To obtain normative values in healthy adults age 20-69 for a CDP system that uses a full-field dynamic visual surround. This is in contrast to the mechanically movable visual surround used in traditional CDP systems. METHODS: Fifty healthy adults divided into five age groups were tested during the three test protocols for CDP: sensory organization test (SOT), motor control test (MCT), and adaptation test (ADT). Outcomes were compared across age groups and to published normative CDP values. Repeatability was assessed in five different subjects (23-30 years old) on 2 days, 1 week apart. RESULTS: Most outcomes were comparable to published norms with notable differences in SOT condition 4 and ADT. SOT composite and conditions 4-6, all MCT translations, and ADT toes up showed moderate to good repeatability (r = 0.60 to 0.99). Age group and gender differences were not substantial. CONCLUSIONS: Some but not all CDP outcomes with a virtual visual environment were comparable to published norms. The differences are likely related to the virtual surround having a more compelling effect on balance even in conditions with a stationary surround.