Cell division modulates prion accumulation in cultured cells - PubMed (original) (raw)

Cell division modulates prion accumulation in cultured cells

Sina Ghaemmaghami et al. Proc Natl Acad Sci U S A. 2007.

Abstract

The phenotypic effect of prions on host cells is influenced by the physical properties of the prion strain and its level of accumulation. In mammalian cell cultures, prion accumulation is determined by the interplay between de novo prion formation, catabolism, cell division, and horizontal cell-to-cell transmission. Understanding this dynamic enables the analytical modeling of protein-based heritability and infectivity. Here, we quantitatively measured these competing effects in a subline of neuroblastoma (N2a) cells and propose a concordant reaction mechanism to explain the kinetics of prion propagation. Our results show that cell division leads to a predictable reduction in steady-state prion levels but not to complete clearance. Scrapie-infected N2a cells were capable of accumulating different steady-state levels of prions, dictated partly by the rate of cell division. We also show that prions in this subline of N2a cells are transmitted primarily from mother to daughter cells, rather than horizontal cell-to-cell transmission. We quantitatively modeled our kinetic results based on a mechanism that assumes a subpopulation of prions is capable of self-catalysis, and the levels of this subpopulation reach saturation in fully infected cells. Our results suggest that the apparent effectiveness of antiprion compounds in culture may be strongly influenced by the growth phase of the target cells.

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Conflict of interest statement

Conflict of interest statement: B.C.H.M., F.E.C., and S.B.P. have a financial interest in InPro Biotechnology, Inc.

Figures

Fig. 1.

Fig. 1.

Cell division decreases the intracellular accumulation of PrPSc but not PrPC. (A) Growth curve for ScN2a cells after a 1:10 split in 100-mm plates. (B–D) The change in the accumulation of PrPSc in ScN2a (B and C) and PrPC in N2a (D) as cells progress from a logarithmic growth phase to stationary phase, as detected by Western blot (B) and ELISA (C and D). After lysis, all samples were normalized to 1 mg/ml total protein concentration as determined by the BCA assay (Pierce) before further processing. The plotted ELISA measurements were normalized to measurements at time 0. The dashed lines indicate the background signal level as determined by plating a mock PBS + 1% BSA sample. The error bars represent standard deviations of three replicate measurements. The solid line in C indicates the predicted PrPSc fluctuation based on the limited conversion model and Eq. 8 (

SI Text

).

Fig. 2.

Fig. 2.

Dividing cells are able to maintain a reduced steady-state level of PrPSc. ELISA measurements of PrPSc levels were normalized to measurements at time 0. The dashed line indicates a background signal from mock PBS + 1% BSA. The solid line represents predicted PrPSc levels according to the limited conversion model and Eq. 8 (

SI Text

). Error bars indicate standard deviations of three replicate measurements. Inset shows the experimental design. ScN2a cells were maintained in a perpetual state of cell division for 5 days by repetitive cycles of subconfluent dissociation and replating (see Materials and Methods). The arrows represent the times of dissociation and replating at the indicated ratios. The predicted cell numbers (relative to a confluent culture) during the course of the experiment are also shown.

Fig. 3.

Fig. 3.

In ScN2a cultures, the rate of horizontal cell-to-cell infection is slow relative to the rate of cell division. (A) Fluorescence distribution in the following cell lines, as measured by FACS: (1) N2a cells stably transfected with GFP, (2) a 7-day coculture consisting of a 1:1 ratio of ScN2a-N2aGFP cells, and (3) nontransfected ScN2a cells. The dashed line indicates the gate used to separate “high GFP” cells from “low GFP” cells. (B) After FACS, the fluorescence distribution of the sorted coculture shows that “high GFP” subpopulation (2) was successfully separated from “low GFP” subpopulation (1). (C) PrPSc levels of the sorted subpopulations from 1:1 Sc2Na−N2aGFP mixtures, as determined by ELISA. PrPSc levels from sorted subpopulations are similar, whether the ScN2a + N2aGFP mixtures were cultured independently then mixed before FACS analysis (gray bars) or cocultured for 7 days (black bars). These data indicate that cells descending from N2aGFP do not contain levels of PrPSc above the experimental error of the experiment. As controls, PrPSc levels in 100% ScN2a and N2aGFP cultures (white bars) are plotted. The PrPSc signal in the sorted low GFP population is reduced because of the presence of transfected N2a cells with low GFP fluorescence levels (see profile 1 in A). The PrPSc signal seen in the sorted high GFP population is due to cross-contamination and represents the experimental background because of sorting artifacts.

Fig. 4.

Fig. 4.

Features and predictions of the limited conversion model compared with the simplified heterodimer conversion model. The limited conversion model posits that in fully infected cultures, when the cell accumulates high concentrations of PrPSc, the subpopulation of the PrPSc that is infectious (capable of self-catalysis) reaches saturation levels (Top). This assumption alleviates some of the experimental inconsistencies associated with treating the processes of PrPSc conversion, PrPSc degradation, and cell division as first-order reactions. The model explains the ability of cells in culture to attain stable levels of PrPSc when fully infected (Middle). It also provides an explanation for the ability of cells to achieve different steady-state levels of PrPSc when dividing at different rates (Bottom). In the simulation shown, stationary cells that have achieved a steady-state level of PrPSc are induced to divide at a time indicated by the arrow. In such a scenario, the simplified heterodimer model predicts a complete clearance of PrPSc over time, whereas the limited conversion model predicts a new steady-state level dictated by Eq. 6. The heterodimer model predicts an unstable state when [PrPC] is assumed to remain constant (as has been experimentally shown here). When this constraint is removed, the model is able to predict a stable state.

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