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 fully understood. This is partly because no in vitro model has been available where nontransformed polyploid human cells can propagate.

Some researchers have induced tetraploidy in nontransformed human epithelial cells through generation of binucleated cells by inhibiting cytokinesis^7-9. In this method, however, unnecessary diploid cells must be eliminated by fluorescence-activated cell sorting (FACS) ^7,8 or cloning by limiting dilution ⁹. Because these procedures are laborious and not easy to perform, simpler methods to establish nontransformed tetraploid cells are desired for research in this field.

In the present report, we describe a protocol to establish proliferative tetraploid cells from normal human fibroblasts or telomerase-immortalized human fibroblasts by relatively simple procedures. The procedures use spindle poison demecolcine (DC) to arrest diploid cells in mitosis, and mitotic cells collected by shaking off are further treated with DC. Diploid mitotic cells treated with DC for prolonged time convert to tetraploid G1 cells, and these cells proliferate as tetraploid cells after growth arrest for several days following drug removal. This protocol provides an efficient method for creating a useful model to study the relationship between chromosome instability and the oncogenic potential of polyploid human cells.

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 evaluate cell growth and cell age. PDL can be calculated from the cell number seeded into a dish (N1) and the cell number harvested at the next passaging (N2) using the following formula (X is the initial PDL). PDL = X + (log N1 - log N2) / log 2

2. Induction and Establishment of Tetraploid Cells from Diploid Fibroblasts

1. Induce tetraploidy without shake-off.
   NOTE: Some cell strains (e.g. TIG-1) can become almost completely tetraploid by continuous treatment with demecolcine (DC) as follows.
   1. Passage cells into one or two 100 mm dishes on the day before treatment to induce tetraploidy. Usually, prepare 5 x 10⁵ to 1 x 10⁶ cells per dish for induction of tetraploid cells.
   2. Treat exponentially growing cells with medium containing 0.1 µg/mL DC for 4 days.
   3. Replace DC-containing medium with drug-free medium and incubate cells in a 5% (v/v) CO₂ atmosphere at 37 °C. Cells usually resume proliferation within 1 week.
2. Induce tetraploidy with shake-off.
   NOTE: Most cells become a mixture of diploid and tetraploid populations as a result of the simple treatment with DC described above (2.1). Therefore, some modifications of the procedure to eliminate a diploid population are necessary as follows (Figure 1).
   1. Passage cells into several T75 flasks the day before treatment to induce tetraploidy. Usually, prepare at least 5 x 10⁶ cells (1 x 10⁶ cells per flask) for induction of tetraploid cells.
   2. Treat exponentially growing cells with medium containing 0.1 µg/mL DC for 16 - 18 h to arrest cells in mitosis. The time required to arrest cells in mitosis may depend on target cells and should be determined by preliminary experiments to obtain as many mitotic cells as possible. For example, slower growing cells should be treated with DC for longer time than faster growing cells.
      NOTE: If cells are treated for too long in this step, they might undergo mitotic slippage and adhere to the dish surface, making it impossible to collect mitotic cells by shake-off in the next step.
   3. Collect DC-arrested mitotic cells by the shake-off method, in which loosely adhered mitotic cells are collected by gentle shaking of flasks and washing with medium followed by centrifugation (300 x g, 5 min). Count cell number by an appropriate method in this step.
   4. Reseed collected mitotic cells into 60 mm culture dishes and treat cells with 0.1 µg/mL DC for an additional 3 days. Seed more than 1 x 10⁶ mitotic cells per dish, because less than 10% of cells can survive this treatment.
   5. Replace DC-containing medium with drug-free medium and wait for cells to resume proliferation, which usually occurs within 1 week.
3. Passage cells similarly to original diploid cells.

3. Examination of DNA Ploidy

NOTE: This is a well-established procedure and a technical manual should be referred to for detailed procedures and technical tips.

1. Harvest cells (5 x 10⁵ - 1 x 10⁶) by incubating cells with a solution containing 0.05% trypsin and 0.02% EDTA for 10 min at 37 °C, and neutralize trypsin by medium containing 10% FBS.
2. Centrifuge cells in medium and wash them by resuspending in 5 mL of phosphate-buffered saline (PBS) followed by centrifugation (300 x g, 5 min).
3. Prepare cells for DNA analysis by one of the following two methods
   1. A method for analyzing unfixed cells¹⁰
      NOTE: This method can be performed using a commercially available kit for cell cycle analysis. Samples prepared with this method should be analyzed immediately, because cells are not fixed in the procedure and stained nuclei will become damaged over time.
      1. Wash cells with 40 mM citrate buffer containing 250 mM sucrose. Subsequently treat cells with 250 µL of 0.1% (v/v) Nonidet P40 and 30 µg/mL trypsin in 200 µL of 40 mM citrate buffer containing 250 mM sucrose for 10 min at room temperature.
      2. Add 200 µL of 0.5 µg/mL trypsin inhibitor and 0.1 µg/mL RNase A in 40 mM citrate buffer containing 250 mM sucrose, and incubate cells for 10 min at room temperature.
      3. Add 200 µL of 0.4 mg/mL propidium iodide (PI) in 40mM citrate buffer containing 250 mM sucrose, and incubate cells for 10 min at room temperature to stain cell nuclei.
   2. A method for analyzing fixed cells
      1. Fix cells with 80% ethanol by suspending cells in 1 mL of PBS, adding 4 mL of 100% EtOH dropwise while mixing the cell suspension and leaving it at room temperature for 30 min. Cells in this fixative can be stored at -20 °C for several weeks.
      2. Wash cells by resuspending them in 5 mL of PBS followed by centrifugation (300 x g, 5 min). Treat cells with 0.5 mg/mL RNase A and stain them with 50 µg/mL propidium iodide (PI) (475 µL of 0.5 mg/mL RNase A and 25 µL of 1 mg/mL PI) for 30 min at room temperature.
4. Analyze DNA content of the cells using a flow cytometer with a 488 nm laser and red channel emission filter (long pass > 610 nm). Analyze a reference sample prepared from genuine diploid cells at the same time to assess ploidy of the target cells.
   1. Alternatively, add fluorescence standard beads to assess the fluorescence intensity ratio of diploid cells vs. the beads. Use the 'pulse height vs. pulse width' option of the flow cytometer to discriminate between single tetraploid cells and clumps of diploid cells¹¹.

4. Examination of Chromosome Counts and Karyotype Analysis

NOTE: These are all well-established procedures and a technical manual or the manufacturer's protocols should be referred to for detailed procedures and technical tips.

1. Treat exponentially growing cells with 0.1 µg/mL DC for 4 h to arrest cells in metaphase.
2. Prepare chromosome slides.
   1. Harvest cells (at least 5 x 10⁵ cells) by trypsinization and suspend in medium containing FBS.
   2. Centrifuge cells in medium (300 x g, 5 min) and treat with hypotonic solution (5 mL of 0.075 M KCl) at 37 °C for 10 min.
   3. Fix and suspend cells in Carnoy's fixative (methanol:acetic acid = 3:1).
   4. Spread cells onto a glass slide by dropping a small volume (25 - 35 µL) of cell suspension. Additional steps such as exposure to hot steam might be necessary to improve chromosome spreading.
3. Stain cells for chromosome counts or karyotype analysis or multicolor fluorescence in situ hybridization (mFISH).
   1. Chromosome counts.
      1. Stain cells with 5% Giemsa solution (or 5 µg/mL PI containing 0.5 mg/mL RNase A for fluorescence microscopy) for 15 - 20 min.
      2. Digitally photograph at least 50 metaphase cells.
      3. Count chromosome number per cell using an image analysis software after manual separation of touching and overlapping chromosomes using a photo editing software. Although chromosome number can be scored manually, computerized scoring is recommended.
   2. Karyotype analysis
      NOTE: This analysis should be performed by a trained technician or a professional company.
      1. Stain cells according to a standard G-banding technique¹².
      2. Analyze chromosomes to make standard karyograms¹².
   3. mFISH
      NOTE: This step is optional and should be performed if necessary.
      1. Stain cells using multicolor FISH probes for human chromosomes according to the manufacturer's protocol¹³.
      2. Analyze chromosomes using a fluorescence microscope with suitable filters and a software for mFISH analysis¹³.

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) (Figures...

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