Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Summary

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

Abstract

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Introduction

Polyploidy has been observed not only in specialized tissues of mammalian species but also in a variety of pathological conditions, such as cancer and degenerative diseases. Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues, such as Barrett's esophagus ^1,2 or squamous intraepithelial lesions of the cervix ^3,4, and have been considered to be the source of malignant aneuploid cells in those tissues ^5,6. Although it is suggested that conversion of tetraploid to aneuploid cells could be a crucial event in the early stages of tumorigenesis, the mechanisms involved in this process are not fu....

Protocol

1. Cell Culture

1. Obtain the cells to induce tetraploidy. To date it has been confirmed that this technique can be applied to the human fibroblast cell lines TIG-1, BJ, IMR-90 and telomerase-immortalized TIG-1 (TIG-hT).
2. Grow cells in minimum essential medium with α modification or any other cell culture medium suited for the cell type to be studied supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) by incubating in a 5% (v/v) CO₂ atmosphere at 37 °C. Passage cells every 3 or 4 days not to exceed subconfluent density.
   NOTE: Population doubling level (PDL) should be calculated each time at passaging to e....

Results

In our experience, TIG-1 cells can be made almost completely tetraploid by simple continuous treatment with 0.1 µg/mL DC for 4 days (Figure 2A). In contrast, other fibroblast strains, such as BJ or IMR-90, and TIG-hT cells, became a mixture of diploid and tetraploid populations following the same treatment, and isolation of mitotic cells by the shake-off method is necessary during the course of DC treatment (usually 16 - 18 h after the start of treatment) (Fi.......

Discussion

A major problem in induction of tetraploidy from diploid cells by chemical agents, either by cytokinesis inhibitors or by spindle inhibitors, is that cells often become a mixture of diploid and tetraploid populations, and tetraploid cells must be separated from diploid cells. Most common approaches for isolation of a tetraploid population free of diploid cells use FACS or cloning by limiting dilution. However, these procedures are laborious and not easy to perform. In this report, we present a new protocol to establish p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MEM-α	Sigma-Aldrich	M8042-500ML
Trypsin-EDTA	Sigma-Aldrich	T4174
FBS	Sigma-Aldrich	172012-500ML
Demecolcine solution (10 μg/mL in HBSS)	Sigma-Aldrich	D1925-10ML
BD CycleTES Plus DNA Reagent Kits	BD Biosciences	#340242	For examination of DNA ploidy by flow cytometry
Human chromosome multicolor FISH probe 24XCyte	MetaSystems	#D-0125-060-DI	Specialized filter set and software for mFISH analysis are necessary
Isis imaging system with mFISH  software	MetaSystems		Specialized probe kit is necessary