Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1999-11-22
2001-07-10
Nolan, Patrick J. (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S004000, C435S032000
Reexamination Certificate
active
06258553
ABSTRACT:
BACKGROUND
1. Field of the Invention
The invention relates to methods of measuring apoptosis in cell culture. More specifically, the invention provides a method of measuring apoptosis in cells isolated from a subject that are exposed to a putative apoptosis-inducing substance. Even more specifically, the invention provides a method of determining the relative sensitivity and resistance of cells to a chemotherapeutic substance by measuring the apoptosis-inducing activity of the substance.
2. Background Literature
Apoptosis is distinguished from necrosis, the other well recognized form of cell death. Sudden anoxia, thermal extremes, or chemical toxicity cause necrosis. Whole areas of tissue die after these injuries and individual cells have indistinct cytological appearances and disrupted membranes. Apoptotic cells, on the other hand, are decreased in size compared to their viable counterparts due to decreased cell water and loss of membrane-bound cytoplasmic blebs
7,8
. The nuclei of apoptotic cells are homogeneously condensed and often fragmented. Internucleosomal double-stranded cleavage of nuclear DNA correlates closely with these nuclear morphological changes of apoptosis
8
. Despite nuclear fragmentation and cytoplasmic blebbing, apoptotic cells retain their energy supply for an extended period of time and their plasma membranes remain intact
7,8
. In vivo, apoptosis occurs most commonly in individual cells that are scattered among non-apoptotic, normal neighbors. Specific molecules on the surface of the apoptotic cells leads to their prompt recognition and phagocytosis by macrophages
7,8
. This rapid removal of individual cells makes apoptosis much less apparent than necrosis, in vivo. Most chemotherapeutic agents used to treat acute leukemia induce apoptosis in vitro in leukemic cells lines and freshly isolated leukemic cells
9-17
. Apoptosis has been demonstrated in the blood and bone marrow of patients receiving combined chemotherapy for acute leukemia
18
. Thus, the measurement of apoptosis in vitro should provide a means to assay for chemosensitivity of a purified leukemic cell population.
Using a population of cells, apoptosis can be identified by the cleavage of DNA at internucleosomal sites
8
. This procedure requires DNA extraction, processing, separation by size, and a means of quantifying intact and cleaved DNA. By examining individual cells within a population, the morphological appearance of apoptosis can be discerned by decreased cell size with condensed, often fragmented, nuclei
7
. A more sensitive morphological test for apoptosis is the terminal deoxynucleotidyl transferase (Tdt)-linked labeling of DNA strand ends which gives an extremely intense signal in apoptotic cells as compared to nonapoptotic ones
19
. However, these morphological methods for detecting apoptosis require cytological or histological preparations which must be examined by light microscopy, fluorescence microscopy, or fluorescence-activated cytometry. Furthermore, with each of these methods for detection of apoptosis, only one point in time may be examined per sample that is processed. Thus, it is not possible to perform real time assessments of apoptosis over time.
Chemotherapy of acute non-lymphocytic leukemia (ANLL) is successful in inducing a remission in most patients, but the majority of patients will suffer relapses and eventually succumb to their disease. These leukemias are often more resistant to chemotherapy at the time of relapse than they were at the time of initial diagnosis. This resistance to one or more chemotherapeutic agents results from the emergence of leukemic cells in the relapsed state that either incorporate less of the agent or that have developed an intracellular mechanism to circumvent the effects of the agent. Multiple factors intrinsic to the leukemia cells such as their numbers at diagnosis, specific karyotypic abnormalities
1
and the ability to grow spontaneously in culture
2,3
can be correlated with the incidence of relapse. However, these are not completely accurate predictors of relapse. The sensitivity of the leukemia to chemotherapy also appears to play a role in determining the incidence of relapses. In vitro assays of chemosensitivity have provided a means to estimate resistance to chemotherapy and thereby provide probabilities for both achieving remission and for developing relapse
4
. However, these assays are cumbersome, time consuming and not entirely reliable. Thus, a means to predict more accurately and easily at the time of initial diagnosis which patients will have a relapse is needed. It is also important to identify the chemotherapeutic agents to which an individual's relapsed leukemia has become resistant.
To achieve these aims, the invention provides a newly developed automated assay that can detect the chemosensitivity of an individual patient's leukemic cells in 48 hours or less. This assay determines the amount apoptosis or programmed cell death that a specific concentration of a chemotherapeutic agent will induce in the patient's leukemic cells.
SUMMARY OF THE INVENTION
A method of measuring the apoptosis-inducing activity of a substance in cultured, isolated cells from a subject is provided, comprising: a) obtaining a sample of cells from a subject; b) isolating a single cell suspension from the sample; c) placing the cells in culture conditions; d) exposing the cells in culture to the putative apoptosis-inducing substance; e) incubating the cultured cells; f) measuring in a serial manner the optical densities of the culture to obtain an optical density curve; and g) correlating the slope of a line representing an increase over time in optical density, due to cellular membrane distortion and blebbing, with an increase in apoptotic activity.
The invention also provides a method of determining the anti-leukemic activity of a substance, comprising: a) obtaining a sample of cells from a subject with leukemia; b) isolating a single cell suspension from the sample; c) enriching the sample for leukemic cells by removing non-leukemic cells from the sample; d) placing the enriched leukemic cells in culture; e) exposing a culture of the enriched cells to the substance; f) incubating the cultured cells; g) measuring in a serial manner the optical densities of the culture exposed to the substance; h) measuring in a serial manner the optical densities of a culture of the enriched cells not exposed to the substance; i) subtracting at each serial time point the optical densities of the culture of cells not exposed to the substance from the optical densities of the culture of cells exposed to the substance, so as to obtain the net slope (as defined above) of the serially measured optical densities due to the apoptosis-inducing activity of the . substance; and j) correlating the slope of a net.increase over time in the serially measured optical densities of the cells exposed to the substance with the anti-leukemic activity of the substance.
The invention also provides a method of determining resistance of leukemic cells to an anti-leukemic substance, comprising: a) obtaining a sample of cells from a subject with leukemia; b) isolating a single cell suspension from the sample; c) enriching the sample for leukemic cells by removing non-leukemic cells from the sample; d) placing the enriched leukemia cells in culture; e) exposing a culture of. enriched cells to the substance; f) incubating the cultured cells; g) measuring in a serial manner the optical densities of the culture of enriched cells exposed to the substance; h) measuring in a serial manner the optical densities of a culture of the enriched cells not exposed to the substance; I) subtracting at each serial time point the optical densities of the culture of cells not exposed to the substance from the optical densities of the culture of cells exposed to the substance, so as to obtain a net slope (defined as recited above) of the serially measured optical densities due to the apoptosis-inducing activity of the substance; and j) correlating the absence of a net increase or the
Nolan Patrick J.
Pennie & Edmonds LLP
Vanderbilt University
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