Dataset Information

Transcriptomic and Proteomic Response of HC-04 to hepatocyte growth factor (HGF)

ABSTRACT: The routine study of human malaria liver-stage biology in vitro is hampered by low infection efficiency of human hepatocellular carcinoma (HCC) lines (<0.1%), poor understanding of steady-state HCC biology, and lack of appropriate tools for trace sample analysis. HC-04 is the only HCC that supports complete development of human malaria parasites. We hypothesized that HCCs are in various intermediate stages of the epithelial-mesenchymal transition (EMT) and HC-04s retain epithelial characteristics that permit infection. We developed an novel analytical approach to test this hypothesis viz. the HC-04 response to hepatocyte growth factor (HGF). We used online two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) to quantify protein expression profiles in HC-04 pre-/post-HGF treatment and validated these results by RT-qPCR and microscopy. We successfully increased protein identification efficiency over offline-2D methods by 12-fold using less sample material, allowing robust protein quantification. We observed expected up-regulation and down-regulation of EMT protein markers in response to HGF, but also unexpected cellular responses. HC-04 is thus susceptible to HGF-mediated signaling but to a lesser degree than what we observed for HepG2, a widely used, but poor malaria HCC model. Our analytical approach to understanding the basic biology of HC-04 helps us understand the factors that may influence its utility as a model for malaria liver-stage development. We observed that HC-04 treatment with HGF prior to the addition of Plasmodium falciparum sporozoites did not facilitate cell invasion, arguing for unlinking the effect of HGF on malaria liver stage development from hepatocyte invasion. Finally, our 2D-LC-MS/MS approach and broadly applicable experimental strategy should prove useful in the analysis of various hepatocyte-pathogen interactions, tumor progression and early disease events.

INSTRUMENT(S): 6520 Quadrupole Time-of-Flight LC/MS

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte

DISEASE(S): Plasmodium Falciparum Malaria

SUBMITTER: Dingyin Tao

LAB HEAD: Rhoel R. Dinglasan

PROVIDER: PXD000682 | Pride | 2014-02-17

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	HC042Dthreetechnicalruns.xml	Xml
	HC04HGFhighsolB01.mzid.gz	Mzid
	HC04HGFhighsolB01.mzid_HC04HGFhighsolB01.MGF	Mzid
	HC04HGFhighsolB01.mzid_HC04HGFhighsolB01.pride.mgf.gz	Mzid
	HC04HGFhighsolB01.pride.mztab.gz	Mztab

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Publications

The acute transcriptomic and proteomic response of HC-04 hepatoma cells to hepatocyte growth factor and its implications for Plasmodium falciparum sporozoite invasion.

Tao Dingyin D King Jonas G JG Tweedell Rebecca E RE Jost Philipp J PJ Boddey Justin A JA Dinglasan Rhoel R RR Dinglasan Rhoel R RR

Molecular & cellular proteomics : MCP 20140216 5

The routine study of human malaria liver-stage biology in vitro is hampered by low infection efficiency of human hepatocellular carcinoma (HCC) lines (<0.1%), poor understanding of steady-state HCC biology, and lack of appropriate tools for trace sample analysis. HC-04 is the only HCC that supports complete development of human malaria parasites. We hypothesized that HCCs are in various intermediate stages of the epithelial-mesenchymal transition (EMT) and HC-04s retain epithelial characteristic ...[more]

PMID: 24532842

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Project description:Mueller2015 - Hepatocyte proliferation, T160 phosphorylation of CDK2 This model is described in the article: T160-phosphorylated CDK2 defines threshold for HGF-dependent proliferation in primary hepatocytes. Mueller S, Huard J, Waldow K, Huang X, D'Alessandro LA, Bohl S, Börner K, Grimm D, Klamt S, Klingmüller U, Schilling M. Mol. Syst. Biol. 2015; 11(3): 795 Abstract: Liver regeneration is a tightly controlled process mainly achieved by proliferation of usually quiescent hepatocytes. The specific molecular mechanisms ensuring cell division only in response to proliferative signals such as hepatocyte growth factor (HGF) are not fully understood. Here, we combined quantitative time-resolved analysis of primary mouse hepatocyte proliferation at the single cell and at the population level with mathematical modeling. We showed that numerous G1/S transition components are activated upon hepatocyte isolation whereas DNA replication only occurs upon additional HGF stimulation. In response to HGF, Cyclin:CDK complex formation was increased, p21 rather than p27 was regulated, and Rb expression was enhanced. Quantification of protein levels at the restriction point showed an excess of CDK2 over CDK4 and limiting amounts of the transcription factor E2F-1. Analysis with our mathematical model revealed that T160 phosphorylation of CDK2 correlated best with growth factor-dependent proliferation, which we validated experimentally on both the population and the single cell level. In conclusion, we identified CDK2 phosphorylation as a gate-keeping mechanism to maintain hepatocyte quiescence in the absence of HGF. This model is hosted on BioModels Database and identified by: BIOMD0000000568. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.