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More information on the growth characteristics of tumors may be gleaned by concommitant measure of cell proliferation and cell death (e.g., by apoptosis) parameters than may be gained by either measure alone. This study reports measures of apoptotic index (AI) and proliferation (following bromodeoxyuridine - BrdUrd - incorporation) in a murine tumor model system. Many methods have been described to measure AI by flow cytometry (reviewed by Darzynkiewicz et al.,2,3). We have selected the in situ terminal deoxynucleotidyl transferase (TdT) assay. This is a flow-cytometric adaptation (3) of the immunohistochemical TUNEL (1) assay. The advantages of this particular assay have been described in detail (3). Briefly, the lesions are identifiable at the molecular level; the DNA breaks occur early in the apoptotic process, prior to morphological changes; concomitant measure of DNA content allows apoptosis to be related to the cell's position in the cell cycle and ploidy status, and the reactions may be carried out in ethanol-fixed samples.

In vivo measurements of AI in murine tumors.

There are very few reports in the literature of the flow cytometric measurement of apoptosis in solid tissues and tumors (4,5). We have used the terminal transferase reaction (TdT) to quantitate the rate and extent of apoptosis induction by paclitaxel in the murine mammary carcinoma MCa-4 (5). Tumors were implanted intra-muscularly and allowed to grow to 8mm diameter before injecting the mice i.v. with 40 mg/kg paclitaxel (Baker-Norton Pharmaceuticals). Tumors were excised for analysis from 1-72 hours after paclitaxel, fixed and single cell preparations made. The apoptotic cells were stained with an avidin-biotin-FITC anti-dUTP antibody TdT-mediated procedure (adapted from that described in refs. 3,4). Total DNA content was stained with propidium iodide (PI) and the data were analysed with bivariate PI vs. FITC flow cytometry (Figure 1).

Figure 1: DNA (PI) vs. apoptotic fragments (FITC), visualized using the terminal transferase reaction, for mouse mammary carcinoma MCA-4, 6 hours after treatment with paclitaxel. The boxed area encloses labeled apoptotic nuclei fragments with G1 and sub-G1 DNA contents.

The control apoptotic frequency was 9%. One hour after taxol this had fallen to 2%. The apoptotic frequency then rose to a peak of 14% by 24 hours and then returned to control levels by 72 hours. This response is qualitatively and quantitatively similar to that found by micromorphometric histological methods in this tumor (Figure 2).

Figure 2: Apoptotic index (%) in a murine mammary adenocarcinoma (Mca-4) as a function of time after treatment with 40 mg/kg paclitaxel. The flow-cytometric data give the same magnitude and time -course of apoptosis as observed using micromorphometric methods.

In separate experiments, mice were treated with paclitaxel and at 1,6,15,24, and 72 hours thereafter the animals were pulse-labeled with 60 mg/kg BrdUrd i.p. Tumors were excised immediately and at 1, 3 or 6 hours after BrdUrd and the dynamic kinetics evaluated using bivariate DNA vs. BrdUrd flow cytometry (6). The flow-cytometric analysis showed cell cycle arrest after paclitaxel treatment. This was seen as early as 1 hour after treatment and was maintained for 24 hours. Evidence for the release of the block was seen by 72 hours after paclitaxel. The flow data confirmed an histological analysis of paclitaxel treated tumors which showed a peak of mitotic arrest (36%) at 9 hours after treatment. This was followed by a peak in apoptotic incidence of 20% at 18-24 hours.

The studies made on the MCa-4 mammary carcinoma examining the tumor proliferation kinetics at various times following administration of taxol showed two critical phenomena. The first was a fairly rapid alteration of the kinetics (the duration of G2 and Mitosis (TG2+M) increased twofold, the labeling index (LI) dropped markedly but the duration of S-phase (TS) remained unchanged), as observed by bivariate measurement of the DNA content of BrdUrd labeled cells, that continued for a prolonged period of at least 2.5 cell cycle times. While the alteration itself occurred fairly rapidly, the kinetics of the treated population appeared quite stable, with only a slow return to normal kinetics. Thus it is possible to regard the treated tumor as if it were in a steady state kinetic situation. The second phenomenon was the appearance of an abnormal number of particles with a sub-G1 DNA content (Fig. 3).

Figure 3: DNA content (PI) vs. BrdUrd incorporation (FITC) for tumor MCa-4 6 hours after a BrdUrd pulse label and 24 hours after treatment with paclitaxel. The boxed areas show the BrdUrd-labeled regions from which the kinetic parameters are calculated. BrdUrd-labeled and -unlabeled fragments can clearly be seen with sub-G1 DNA contents (Ap).

These nucleic fragments were both labeled and unlabeled and evidently represent a subpopulation that is dying through an apoptotic-like pathway. Since the labeled fragments must have been synthesizing DNA prior to entering into a death pathway, these data provide both the basis for a method for estimating the cell death rate through that pathway and enhanced information about the proliferation kinetics of a tumor following chemotherapy.


Literature cited:- 

1. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J Cell Biol 1992;119:493-501.

2. Darzynkiewicz Z, Bruno S, Del Biano G, Gorczyca W, Hotz MA, Lassota P & Traganos F. Features of apoptotic cells measured by flow cytometry. Cytometry 1992;13:795-808.

3. Gorczyca W, Gong J & Darzynkiewicz Z. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Research 1993;53:1945-1951.

4. Steck KD, McDonnell TJ & El-Naggar AK. Flow cytometric analysis of apoptosis and BCL-2 in human solid neoplasms. Cytometry 1995;20:154-161.

5. Terry NHA, Milross CG, Patel N & Milas L. Flow-cytometric measurement of apoptosis and tumor cell kinetics following treatment with taxol (abstract). 43rd Annual Meeting of the Radiation Research Society, San Josť, CA, April, 1995.

6. Terry NHA, Milross CG, Patel N, Mason KA, White RA & Milas L. The effect of paclitaxel on the cell cycle kinetics of a murine adenocarcinoma in vivo. The Breast Journal, 3: 99-105, 1997 .


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