Structuredness of the cytoplasmic matrix and Michaelis-Menten constants for the hydrolysis of FDA during the cell cycle in Chinese hamster ovary cells

Summary.-The reaction rates of enzymes hydrolysing fluorescein diacetate have been studied in populations of intact tissue-culture EMT6, cells using flow cytofluorimetric techniques. It was found that the activity of these enzymes increased in plateau phases and that this correlated inversely with plating efficiency. Highly abnormal substrate-dependent reaction velocity kinetics were found in 14-, 21-, 28-and 35-day cultures. IN previous studies (Watson and Chambers, 1977) it has been shown that an enriched clonogenic fraction of EMT6 cells grown in vivo and separated by their ability to stick to plastic (Twentyman and Watson, 1977) has higher RNA levels than those cells which do not stick to plastic and which have a low plating efficiency. However, as the RNA distributions of the high and low clonogenic fractions overlapped, work was undertaken to find a second biochemical parameter which could be assayed simultaneously with RNA, in order to obtain a better discrimination between the 2 populations. Enzymes hydrolysing fluorescein diacetate can be assayed in populations of intact cells in suspension, using flow cytofluorimetric techniques and it was decided to study these enzymes to see whether hydrolysis rates could be utilized in conjunction with RNA measurements. The work presented in this communication compares the enzyme activities in populations of individual EMT6/M/CC cells during exponential growth and at 5 stages during the plateau phase and the results are related to changes in plating efficiency. MATERIALS AND METHODSEMT6/M/CC cells.-These are a variant of a mouse mammary tumour line (EMT6) (Rockwell, Kallman and Fajardo, 1972) which has been maintained in tissue culture for over 3 years in our laboratories. The preparation of single-cell suspensions and growth kinetic data have been reported previously by Twentyman et al. (1975). For these studies, cells were assayed during exponential growth and during the plateau phase at 7, 14, 21, 28 and 35 days after seeding the monolayer. Immediately before the enzyme assay, the monolayers were trypsinized and the cells were resuspended in medium at a concentration of 1-2 X 106/ml.Five separate experiments were carried out over a number of weeks. At each run, exponentially growing cells were assayed in parallel with one of the 5 ages of plateau phase. The culture medium was changed daily from Day 3 onwards in all plateauphase flasks. Plating efficiencies were also carried out in conjunction with the enzyme studies.Enzyme reaction.-The hydrolysis of fluorescein diacetate (FDA) is catalysed by a variety of hydrolytic enzymes, including lipase, acylase and a-and y-chymotrypsin (Guilbault and Kramer, 1966). We shall refer to these enzymes collectively as esterase. Activity

Section: )Iscussionmentioning

confidence: 99%

Different esterase activities of exponential and plateau phases of EMT6 cells monitored by flow cytofluorimetry

Watson¹,

Workman²,

Chambers³

1978

“…Changes in the SCM are measured on cell suspensions with the technique of fluorescence polarization in a fluorescence spectrophotometer of high sensitivity (Cercek, Cercek and Ockey, 1973). These measurements yield data on average changes in the SCM over whole cell populations, and cannot yield estimates of the percentages of cells which change their SCM on stimulation.…”

mentioning

confidence: 99%

Changes in the structuredness of cytoplasmic matrix (SCM) in human lymphocytes induced by PHA and cancer basic protein as measured in single cells

Cercek¹,

Cercek²

1976

“…Granulocytic cells proliferating in a long term (2-10 week) culture system developed by Dexter et al (1974) (Cercek et al, 1973c). We estimate the values of P in these experiments to be accurate to within 500.…”

Section: Preparation Of Test Cellsmentioning

confidence: 87%

“…accompanied by a decrease in SCM. This biophysical change affects the rate constants of enzyme reactions in the cell and it is therefore to be expected that changes in SCM could be important in the regulation of proliferative processes (Cercek and Cercek, 1973a;Cercek, Cercek and Ockey, ] 973c).…”

Section: Preparation Of Test Cellsmentioning

confidence: 99%

“…This organization is the result of physical interactions between macromolecules such as proteins, water molecules and solutes (Ling, 1972). Perturbations of these interactionis result in a change in SCM Cercek, 1972a, b, 1973a, b;Cercek, Cercek and Ockey, 1973c;Cercek, Cercek and Garrett, 1973d (Lasalvia, Garcia-Giralt and Macieiro-Coelho, 1970;Houck et al, 1971). …”

mentioning

confidence: 99%

See 1 more Smart Citation

Inhibitors of Haemopoietic Cell Proliferation?: Specificity of Action Within the Haemopoietic System

Lord¹,

Cercek²,

Cercek³

et al. 1974

Summary.-The specificity of action of mature blood cell extracts on their own progenitor cells was investigated by measuring their effects on the structuredness of the cytoplasmic matrix (SCM) using the technique of fluorescence polarization.Changes in SCM induced by the various extracts are probably closely related to the proliferation state of the cells.Saline extracts of lymphocytes, granulocytes and erythrocytes (LNE, GCE and RCE respectively) have been partially purified by ultrafiltration into selected molecular weight ranges and each tested against proliferative populations of lymphoid, granulocytic and erythroid cells. In all cases, complete specificity of effect on SCM was found, LNEs affecting only lymphoid cell populations, GCEs affecting only the granulocytic cell populations and RCEs affecting only erythroid cells. In each case, with the possible exception of the RCEs, the active fractions reside in the molecular weight ranges reported in the literature inhibitory properties.