Biochemical studies on mast cell tumours in culture

The major part of the life cycle of actively growing and dividing cells, often referred to as interphase, is the period during which all cell components are duplicated so that during the brief mitotic period, the cell can split into two similar daughters. This thesis describes an investigation into the processes of cell component duplication during the interphase of cultured neoplastic mast cells.
Chapter 1 sets out the concept of partitioning the cell cycle into 4 phases: G1, S(DNA synthesis), G2 and M(mitosis). It contains a survey of techniques used to study the life cycle of cells, concentrating especially on the synchronization of mammalian cells in culture. Chapter 2 describes the general experimental techniques used throughout the study.
Chapter 3 describes the development of gradient centrifugation as a method (i) to produce synchronously-growing cells and (ii) to obtain large numbers of suspension culture cells at specific stages of the cell cycle.
Cells collected from an exponentially-growing culture were centrifuged on a Ficoli gradient. Fractions from various areas of the gradient incubated in fresh growth medium grew synchronously as judged by cell number, relative volume, mitotic index and thymidine incorporation. Thus small cells from the tipper region of the gradient had growth characteristics expected of G1 cells while larger cells from the lower region behaved In the manner expected of G2 cells. Cells taken after mixing the whole gradient showed an exponential/growth curve typical of unsynchronized cells. Thus gradient centrifugation does not subject cells to adverse physiological effects.
Analysis of cells pre-labelled with thymidine and separated on the gradient confirmed that cells near the top were in G1, cells near the bottom in G2, and, in addition, showed that those in the middle were in the period of DNA synthesis (S).
Limitations of 'conventional' gradient centrifugation, such as poor resolution at the S/G2 region and low yield, were overcome by using a slow-speed zonal rotor.
Chapter 4 describes how gradient centrifugation has been applied to the study of the synthesis of phospholipids in relation to that of protein, RNA and DNA during the cell cycle.
Analysis of cells pre-labelled with appropriate precursor and separated by conventional gradient centrifugation showed that protein, RNA, and phospholipid synthesis were continuous throughout the cycle and that the rates of synthesis began to increase already during G1. The pattern of phospholipid degradation followed that of synthesis.
Analysis of pre-labelled cells separated by zonal centrifugation confirmed the results obtained by conventional centrifugation and in addition showed that the rates of protein, RNA and phospholipid synthesis reached a maximum in late S, decreasing again during G2. The net amounts of protein, RNA and phospholipid, unlike that of DNA which increased relatively sharply, were found to increase continuously throughout the intermitotic period.
These results show that phospholipid and macromolecular synthesis, and possibly membrane construction, are controlled by a mechanism other than gene dosage.
In Chapter 5, the work on the synthesis of macromolecules is extended by investigating changes in specific proteins, i.e. enzymes. The enzymes chosen were representative of different cell components so that an indication of duplication of intracellular organelles might be obtained.
Two soluble cytoplasmic enzymes (lactate dehydrogenase and glucose-6-phosphate dehydrogenase), a microsomal enzyme (NADPH cytochrome c reductase) and two inner mitochondrial membrane enzymes (cytochrome c oxidase and succinate cytochrome c reductase) were found to show a rather similar variation in concentration (activity/cell) during the cycle, namely an increase starting in G1, continuing through S and reaching a maximum in late S/G2. Thus they followed a pattern generally resembling net protein synthesis. Under certain circumstances, lactate dehydrogenase showed fluctuations superimposed upon a steady increase.
A soluble enzyme of the mitochondrial matrix, glutamate dehydrogenase, showed a somewhat different pattern, the level remaining constant curing G1 and increasing only after DNA synthesis had started.
Studies with fluorescent probes showed that the percentage of mitochondrial-electron-transport-protein relative to total protein remained constant during the cell cycle.
It is concluded that the development during the cell cycle of intracellular structures such as mitochondrial membranes and microsomal membranes probably come under the same control (which is not that of gene dosage) but that glutamate dehydrogenase, located in the mitochondrial matrix, may be subject to a different mechanism.
The scope of future work is discussed.