Una primera aproximación a la categoría constelación de lucha

There are several methods which can be used to assess proliferation characteristics, each having advantages and disadvantages. These can be subclassified into state and rate measurements. The former reflecting the percentage of a proliferation associated population at any particular time and the latter representing measurements in which the cell cycle transit time is calculated to allow estimation of a doubling time.

1.19.1 State measurements

1.19.1.1 The labelling Index

The LI is defined as the number of cells that take up a DNA precursor. The proportion of cells. Traditionally tritiated thymidine was able to estimate the number of cells actively involved in the S phase of the cell cycle and calculate the thymidine-labelling index (TLI) as a marker of proliferation (Taylor J.H., Woods P.S. et al. 1957). This was achieved by pulse labelling of cells with tritiated thymoidine. The percentage of the population labelled was given to be the LI. However, Several assumptions were made. Firstly, the free avaliability of the label to the cells. Secondly, active uptake of the label to the cells and thirdly, efficient detection of the incorporated radioactivity.

1.19.1.2 The Growth Fraction

The growth fraction (Gf) is the proportion of cells in a tumour that are proliferating (in the cell cycle). Direct measurement is difficult because of the uncertainty of defining which cells are in the cell cycle. At present it is considered that that Ki 67 labelling is the closest measurement of the Gf. If Tc and Tpot are known the G f can be calculated from:

Gf=Logp2xTc Tpot

With the advent of novel monoclonal and polyclonal antibody staining systems alternative methods to equate the Gf have been developed such as the Ki-67 (Gerdes, Schwab et al. 1993) antibody or PCNA (Miyachi K, Fritzler et al. 1987).

1.19.1.3 Ki67 antibody staining

The Ki67 antigen is expressed in all stages of the cell cycle except GO (Sasaki, Murakami et al. 1987). There is some variation. In G1 it is expressed in the nucleoli, during S and G2 there is increased intensity with nucleoplasmic distribution and the peak activity is in M where the antigen is chromation associated. Recent evidence has

demonstrated that that its expression may be an absolute requirement for cell proliferation (Duchrow, Gerdes et al. 1994). Although its function and role has not been totally elucidated, it may have a role in the nuclear matrix, condensation of chromosomes or the breakdown of the nuclear envelope prior to mitosis (Schluter, Ducrow et al. 1993). The Ki67 gene is localised on chromosome 10 and encodes a doublet protein of 345 and 395 kDa.

The best known antibody for detecting Ki 67 was the IgG l murine monoclonal antibody derived from mice immunised with extracts from a Hodgkin’s disease cell line L428 (Gerdes, Schwab et al. 1993). However this was hampered by the necessity to use frozen sections thought to be due to the fragility of the Ki67 epitope. More recently, a new series of antibodies has been developed by immunising mice using protein sequences obtained from the central repeat of the Ki67 gene of E. Coli (Key and Becker 1993). As a result the MIB 1 antibody has been developed that has been shown to recognise the Ki67 antigen in formalin fixed paraffin embedded tissue sections after antigen retrieval techniques (Cattoretti, Pileri et al. 1993).

1.19.2 Rate measurements

1.19.2.1 The Volume Doubling Time

The volume doubling time (Td) represents the time interval in which a tumour doubles in volume and this measurement is therefore subject to cell loss, the growth fraction as well as any host cells contained within the tumour mass. It can be derived from: i) Serial sacrifice of animals and weighing the tumours, ii) Sequential measurements with callipers and the use of appropriate formulae for converting linear dimensions to volume, iii) Displacement of fluid giving a volume measurement.

Most tumours reduce growth rate with increasing size and represent a Gomperzian growth curve, ie and exponential growth with an exponential slowing superimposed with increasing age. The Td for normal tissues is infinitely long since cell production

and loss are perfectly matched. The Td of tumours varies between one day in fast rodent tumours to many months in slowly growing human tumours.

1.19.2.2 The Potential Doubting Time

The potential doubling time (Tpot) is a “rate measurement” and is defined as the time within which a cell population would double its number if cell loss did not occur. Thus, if all of the cells were proliferating (GF=1) and there was no cell loss then the Tpot would equal the Tc. It is a good measure of cell proliferation of a tumour because it takes into account the Tc and the GF.

Tpot is determined from an estimate of the proportion of the cells in any phases of the active cell cycle and the duration of that phase. They are usually calculated from either the mitotic index and the duration of mitosis or more commonly from the labelling index (LI) ie the proportion of cells which take up a DNA precursor and the duration of S phase. DNA precursors traditionally included tritiated thymidine which was analysed by high resolution autoradiography but now bromodeoxyuridine is utilised more commonly and analysed by flow cytometry.

T pot is calculated from Tpot=A, x Ts/L I

X is applied because of the nonlinear distribution of cells in the cell cycle such that on average there are twice as many cells in the postmitotic phase G1 than in the immediately G2 premitotic phase (Steel and Lamerton 1966).

To apply this formula the duration of the appropriate phase is required. For mitosis this was obtained by strathmokinetic techniques. Spindle poisons such as colcemid are use to block mitosis and the time taken for the metaphases to double is the mitotic duration. Similarly, repeated injections of tritiated thymidine can be given to calculate phase duration. The advantage of the bromodeoxyuridine and flow cytometric method is that S-phase duration can be calculated from one biopsy using the relative movement method (Begg, McNally et al. 1985).

1.19.2.3 Cell Cycle Time

The cell cycle time (Tc) is the time interval within which one cell completes a mitotic cell cycle, i.e. from birth at mitosis to eventual splitting to two progeny at the next mitotis having passed sequentially through G l, S and G2 phases o f the cell cycle. Traditionally this has been obtained with the percent labelled mitosis (PLM) method. After injection of tritiated thymidine, multiple biopsies, at time intervals, are taken and the amount of radiolabelled mitosis in each sample was determined. Cell cycle times can be calculated from the plot (Figure 1.24) Variation in phase length between cells prevent the curves from being idealised as shown and results in less distinct curves with dampening in successive cell cycles. Computer assisted analysis is, therefore, required.

Tm

Hrs after tritiated thym idine injection