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List of tables and figures

Table 5.1.1. Key to Focus Categorisation

Table 5.1.2. X-ray induced and spontaneous foci Table 5.1.3. Alpha - particle induced foci

Figure one: Photographs of an X-ray induced (+) focus

Figure two: Photographs of an alpha - particle induced (+) focus Figure three: Photographs of an X-ray induced (X/+) focus Figure four: Photographs of an X-ray induced (X/+) focus Figure five: Photographs of an X-ray induced (X) focus Figure six: Photographs of an X-ray induced (-) focus

The C3H10TVS assay is extensively used as a system to assess the carcinogenic properties of a variety of agents including chemicals and radiation. It is the loss of

contact inhibition of the cells when they become transformed that makes the system

particularly attractive. This loss of contact inhibition is observed as a focus of cells

which grow in layers. Although the foci are easily distinguished against a monolayer of contact - inhibited cells, the decision as to what constitutes a positively transformed focus is not as easily resolved. Positively transformed in vitro is considered to be equivalent to tumourigenic in vivo, as one cannot truly apply the term tumourigenic

to these foci until they have proved capable of producing tumours. The foci produced

in the transformation assay were originally classified into three types, designated types

I, II and III, by Reznikoff ei ah in their publication in 1973. Type I foci, composed

of tightly packed cells are not scored as malignantly transformed, since foci isolated

by Reznikoff et al. failed to produce tumours in C3H mice. Type II foci show considerable piling-up of cells into virtually opaque multilayers with criss-crossing of cells not pronounced. The third category of focus, type III consists of multilayered criss-crossing arrays of densely stained fibroblastic cells. Type II and III are classified as malignantly transformed, with fifty percent of type II and eighty five percent of type III producing tumours in C3H mice, as reported by Reznikoff et al.

Not all foci in the C3H10T14 assay fall easily into one of these categories. A

wide range of foci are usually observed with some or all of the above characteristics

and classifying the foci as I, II or III often disguises borderline cases. These borderline

foci can create significant differences in the data obtained in different laboratories, depending on the manner of scoring of the foci and the criteria deemed most important

for the distinction between positively and negatively transformed foci. A catalogue

illustrating the range of foci observed and the classification attributed to the foci has been prepared as part of the collaboration project described in chapter three (copy available from any of the authors).

The most definitive test of positively transformed cells in vitro is the ability

of the cells to produce tumours in vivo. However it is not feasible to isolate and test

all the transformed foci produced in a typical C3H10TV2 assay. Several authors have

isolated sample foci and tested the tumourigenicity (see section 5.2). In this thesis, 5.1.2

data on the isolation and examination of twenty seven X-ray induced foci, one spontaneous focus and sixty alpha-particle induced foci are presented. The isolation

and classification of these foci are described in the following sections.

Focus isolation

Foci isolated, expanded and developed as individual cell lines for use in the

majority of the work in this thesis were produced after irradiation with X-rays or

alpha-particles. Parent C3H10T14 cells were exposed to 5 Gy X-rays or 1 Gy alpha-

particles using the radiation sources as described in chapter two. A standard

transformation assay was then set up (see chapter two) but the cultures were incubated

for ten weeks instead of the standard six weeks adopted by many laboratories or the four weeks postconfluence incubation adopted by the European collaborative project

(details in chapter three). The longer incubation period allowed the foci to grow to a

size which made the isolation of the foci more feasible. Foci to be isolated, were

photographed the day before isolation, while still in culture. Isolation of the focus cells

was accomplished by scraping approximately half of the focus, using a cell scraper

into a small volume of medium which was then aspirated into a separate tissue culture

flask (containing growth medium). The cells were left to attach and grow and subsequently subcultured into larger tissue culture flasks for further growth to cell numbers which allowed frozen stocks of each focus to be established as described in

chapter two. The remaining culture, from which the focus was originally isolated was

stained with a giemsa stain (described in chapter two) and the focus classified using

the criteria described below. The area of the focus remaining on the culture flask was measured using a grid divided into squares of nine square millimetres in area and the

total area of the focus estimated using the same grid. The percentage focus remaining

was then calculated. The alpha-particle induced foci were isolated in two batches, with a week between batches while the X-ray induced foci were isolated together from a single experiment.

Focus classification

the stained remainder of the focus and classified it using the criteria of Reznikoff et al. (1973), but also including a new classification (type X) described below. Foci were again categorised as part of a collaborative project with four other laboratories. The project began in 1990 with laboratories in Berkeley and Harwell in the United Kingdom, Milan and Rome in Italy and Munich and Gottingen in Germany (details

in chapter three) working together to standardise the C3H10T16 assay for use by

collaborating laboratories examining the transforming effects of low doses of radiation. One especially important aim of the project was to standardise the criteria for categorisation of foci. The foci tabulated below were examined, using the stained remainder of the focus and the photographs taken prior to focus isolation, at meetings

of the Berkeley, Milan, Rome, Munich and Gottingen laboratories (eight to ten people

examined the foci). These laboratories have adopted the criteria of Reznikoff et ai,

with a few modifications. No distinction is made between type II and type III foci and

the most important criteria for a positively transformed focus is the presence of criss­ crossing cells. Thus heavily piled-up foci with even a few cells criss-crossing are regarded as positively transformed while a focus presenting piled-up cells only is deemed to be negatively transformed. A new category of focus has been identified in

the process of this collaboration which is designated type X Type X foci present as

long, flowing, fibrous sheets of cells which may exhibit piling up of the cells along

the strands but do not display criss-crossing of cells. This morphology is occasionally

seen in conjunction with criss-crossing in a positively transformed focus.

Table 5.1.1 summarises the various categories into which the foci were placed. Tables 5.1.2 and 5.1.3 list the X-ray induced foci, spontaneous focus and alpha-particle induced foci with the corresponding classifications, details of the percentage focus stained and examined microscopically, as well as comments on the foci by the author.

Figures one to six show photographs of the stained remainder of a number of the foci

from different categories.

It is evident from tables 5.1.2 and 5.1.3 that the area of the X-ray induced foci

ranged from 54mm^ to 450mm^ while that of the alpha-particle induced foci ranged from 18mm^ to 639mm^ with the majority of the foci having areas in the lower half of these ranges. The percentage focus remaining on the culture flask and stained was

up to ninety percent with most foci having below fifty percent stained. Thus in most cases over half of the focus area was removed to develop the cell line and less than

half of the focus available to be examined by routine focus classification criteria.

Comparison of the author’s score for the foci with that of the European collaborative group reveals about fifty percent agreement for the X-ray induced foci and about

seventy percent for the alpha-particle induced foci. In most cases of disagreement

between the scores a higher proportion of foci were scored as (+) by the collaborative group and placed in other categories by the author. In a number of cases there were

additional foci on the culture flasks to those foci isolated, and these foci mostly

resembled the focus from which cells were isolated.

All of the isolated foci described below have been tested for the ability to

induce tumours in C3H mice. A selection of foci from the various categories have

been further tested for the growth characteristics, chromosome complement and ability

to reconstruct foci, results of which are presented in the following sections of this

Table 5.1.1. Key to Focus Categorisation.

Focus Category Description

+ Transformed focus, definite type II or III with criss-crossing arrays of cells

- Not transformed

X Flowing fibrous sheaths of cells, cells may be piled up but are not criss-crossed X/+ Mixed characteristics of type (X) and (+) focus categories

Additional terms used to describe the foci

Term Meaning

I Not transformed, focus of tightly packed cells

II Transformed focus, several layers of cells, criss-crossing of cells not pronounced III Transformed focus, several layers of densely stained, criss-crossing arrays of cells ? Focus difficult to score, category undecided

The table outlines the categories (+), (X/+), (X) and (-) used for scoring the foci listed in the following tables as well as additional terms used in the text to describe the foci. The terms I, II and III refer to the categories originally outlined by Reznikoff et al. (1973), while the categories (+), (X/+), (X) and (-) refer to the categories used for foci examined by the European collaborative group as described in the text.

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Figure two: Photographs of the alpha -particle induced focus a l l . Lines under the

photographs represent one millimetre as determined using photographs of a 1mm Objective

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Figure four; Photographs of the X-ray induced focus X4. Lines under the photographs represent one millimetre as determined using photographs of a 1mm Objective micrometer (Nikon). The collaborative consensus score for this focus was (X/+).

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