Vibrational Spectroscopy of Viable, Paired Tumorigenic and Non- Tumorigenic Cells

Infrared absorption of two pairs of non-tumorigenic and tumorigenic cells suspended in phosphate buffered saline have been measured. Suspensions of cells with several different growth cycle distributions were measured. The effect of both growth cycle and tumorigenity on the infrared absorption spectrum will be presented. For example, changes in absorption in the phosphate absorption region were observed for suspensions with different cell cycle distributions. We will discuss the biochemistry which may cause these changes. We have found that spectra of isolated nuclei allow the DNA spectra to be studied, without the confounding influence of RNA. Therefore, the measurement of isolated nuclei may represent a method of detecting changes in DNA architecture with cell cycle. As part of this exploratory study we have also examined the variation in spectra with cell type and compared epithelial cells with fibroblast cells. Very little change was observed. Similarly we saw very little change in the spectra of tumorigenic and non-tumorigenic cells harvested with similar cell cycle distributions. Changes in the spectra were observed when rapidly growing tumorigenic cells were compared to slowly replicating nontumorigenic cells. INTRODUCTION IR and Raman spectroscopic measurements can provide detailed molecular information. Consequently, these spectroscopies have the potential to generate important biochemical information for tissue diagnosis. Some of the medical applications of vibrational spectroscopy currently under investigation include characterization of atherosclerosis (plaque in arteries), and detection of cancer. METHODS Biochemical components of cells were obtained and measured in aqueous media. Type I "highly polymerized" calf thymus DNA (Sigma) was dissolved in TE buffer (pH 8.02, 10mM TRIS, 1mM EDTA). Type IV calf liver RNA was also dissolved in TE buffer. L-α-phosphotidylcholine and Lα-phosphotidylethanolamine were dissolved in a buffer consisting of 0.01 M, pH 7.3 TRIS, 0.1 M NaCl and 0.02 M sodium azide. Albumin was measured in a pH 7 phosphate buffer and lysozyme was measured in pH 3.8 sodium citrate buffer. Four types of cells derived from rat embryo fibroblast (REF) cells were used. M1 and Rat1 cells are immortalized but non-tumorigenic derivatives of REF cells. MR1 cells are a tumorigenic derivative of M1 cells: specifically, these cells were transfected with the point-mutated T24Ha-ras-oncogene. Similarly, Rat1-T1 cells are a tumorigenic derivative of Rat1 cells obtained by a stable transfection with the same mutant h-ras oncogene. Cells were cultured as monolayers in standard tissue culture flasks using Dulbecco’s modified eagle’s medium containing 4.5 g/l D-glucose, 5% (v/v) fetal calf serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Biomedical Vibrational Spectroscopy II, Anita Mahadevan-Jansen, Henry H. Mantsch, Gerwin J. Puppels, Editors, Proceedings of SPIE Vol. 4614 (2002) © 2002 SPIE · 1605-7422/02/$15.00 109 Two types of epithelial cells were also measured. AT3.1 and AT6.1 are androgen-independent malignant rat prostate carcinoma cells kindly supplied by Dr. Rinker-Schaeffer of the University of Chicago. Cells were cultured as monolayers in α-MEM (Invitrogen) containing 10% (V:V) fortified calf serum (Hyclone Laboratories) and antibiotics (50 μg/ml streptomycin and 50 U/ml penicillin, Invitrogen). Cell suspensions were obtained from monolayer cultures by treatment for 10 minutes with 0.25% trypsin in a Dulbecco’s phosphate buffered saline. The cell suspensions were subsequently centrifuged to obtain a cell pellet; subsequently, phosphate buffered saline (PBS) was added to the cell pellet to obtain a cell suspension with approximately 10 cells/ml. The cell cycle distribution of the cells was determined by flow cytometric DNA content analysis. Nuclei were prepared from cell suspensions using a hypotonic lysis and sucrose gradient procedure. Briefly, a cell suspension in media was centrifuged (1000 rpm x 10 minutes) and the pellet was resuspended in a hypotonic lysis buffer (HLB:10 mM Tris, 5 mM KCl, 1.5 mM MgCl2, pH 7.9) for 10 minutes on ice then passed 12 times through a 22-guage needle to lyse the cells. The nuclei suspension was layered on top of a sucrose cushion (800 mM sucrose in HLB) then centrifuged (5000 rpm x 10 minutes). The nuclei pellet was resuspended in sucrose buffer (250 mM sucrose, 10 mM Tris, 3.3 mM MgCl2, pH 7.9), centrifuged at low speed, and the pellet resuspended in sucrose buffer. This nuclei suspension was then layered on top of another sucrose cushion (350 mM sucrose, 0.5 mM MgCl2) and spun again at high speed. This final nuclei pellet was washed twice with phosphate-buffered saline (PBS) using low-speed centrifugation then resuspended in ice-cold PBS for analysis. Infrared absorption of cells in PBS and of nuclei in PBS was measured on a Mattson Cygnus-100 FTIR with a DTGS detector. The cell suspension was loaded into a 50 micron thick space between two BaFl2 windows. The absorption was calculated as –log(Ic/IPBS), where Ic is the intensity measured when the sample cell was loaded with cells and IPBS is the intensity measured when the sample cell was loaded with saline. Each intensity measurement consisted of 200 scans at 2 cm-1 resolution. For each experiment alternating measurements of cells and PBS were made. Typically two measurements of saline, two measurements of cells, two measurements of saline, etc. For analysis the spectra were scaled to have an absorbance of 0.075 at 1400 cm-1. No baseline correction was performed accept were specifically mentioned in the results section.