**2.8 Study of cell distribution through the cell cycle**

Alterations of the quantitative distribution through the cell cycle were also investigated. The distribution of tumor cells through the cell cycle was studied by flow DNA-cytofluorimetry on ICP-22 cytofluorimeter.

The BSH distribution was investigated in B-16 melanoma bearing male C57Bl/6 mice. The mice had a body weight (BW) of 17-20 g, the tumor was subcutaneously transplanted. BSH was administrated intraperitoneously. 2 dose groups were injected with 50 and 150 mg/kg body weight on 9-11-th day after subcutaneous tumor inoculation. In 3, 12 and 24 hours after administration tumors were excised (3 samples per time point). Tumor samples were used for preparation of cell nuclei suspension. This suspension was dyed with mixture of ethidium bromide and mitramicin (1:1).

### **2.9 Assessment of tumor growth**

During experiments with transplanted tumors three reciprocally perpendicular tumor's diameters were daily measured and evaluated tumors volumes taking tumor's shape as ellipse:

$$\mathbf{V} = (\pi/6). \text{ d}\_1 \text{ d}\_2. \text{ d}\_3 \tag{1}$$

Where V- tumor volume, d1, d2, d3 – linear dimensions of ellipsoid, cm

For every temporary point tumor volume was normalized to it's volume at the beginning of exposure (V0) and then V1/V0 against time from the beginning of exposure curves were constructed depending on every dose D and for every concrete mouse at adequate control for it (D=0). The parameters of tumor growth delay (Td) and of time of tumor's volume duplication were evaluated from these curves as well as tumor growth index (2).

$$\text{TGI} = \text{S}\_{\text{E}} / \text{S}\_{\text{K}} \tag{2}$$

where: SE. – area under tumor growth curve of experimental group, Sк – area under tumor growth curve of experimental group.

Area under tumor growth curve was calculated as following:

$$S = \sum\_{i=1}^{n-1} \frac{V\_i + V\_{i+1}}{2} t\_i \tag{3}$$

where Vi - tumor volume at ith measurement,

n – number of measurements,

ti – period between nearby measurements.
