Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-07-06
2001-08-21
Zitomer, Stephanie W. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S244000, C435S366000, C435S375000, C435S377000, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06277575
ABSTRACT:
The present invention relates, in general, to the determining of a reorganization of the nucleus during tumor suppression and the consequences resulting therefrom both at the diagnostic and at the therapeutic, in particular anticancer therapy, level.
An increasing number of signs suggest that the regulation of the expression of genes, whether regarding their activation or their silencing, occurs in two stages in which a compartmentalization of the chromosomes in the nucleus before transcription is first of all observed.
The present invention has demonstrated the fact that, in cancerous tissues, this compartmentalization occurred differently compared with a normal cell, which could constitute a diagnostic tool.
It has been possible to carry out this observation in particular by studying the mechanism of the p21WAF1 gene and its behavior during the activation which contributes to the suppression of the tumorigenic process.
It has been possible for this phenomenon to be demonstrated in particular by means of an improvement in the DNase I method which made it possible to monitor this process.
The DNase I method is based on the observation that the chromatin active in transcription is more particularly sensitive to the action of nucleases. It contains, in particular, sites which are hypersensitive to DNase I (these are relatively short DNA segments which are very sensitive to a large number of cleaving agents). These nucleosome-free DNA segments are associated with various functions in the expression of the gene. Such “open chromatin regions” may allow the binding of specific factors and possess major regulatory functions; it is said in this case that the labeled DNA is the exposed DNA.
It has therefore been possible to study, by means of this method, the possible relationships between the transcription of genes and their hypersensitivity to DNase I in situ, especially as a marker allowing analysis of the organization of the overall genome in cancer phenomena.
The general method which will be called hereinafter “DNase method” is described in particular in published American Patent U.S. Pat. No. 5,264,343, this document is cited here by way of explicit reference and the information which it contains will not be repeated in greater detail. Reference may also be made to the publication by Puck et al. (Puck T. T., Bartholdi M., Krystosek A., Johnson R. and M. Haag. Somatic Cell and Molec. Genetics 17, 489-503, 1991).
However, the method described in the patent in question does not allow analysis of tissues fixed by the formalin and paraffin method, that is to say tissues obtained by the method used in most anatomical pathology laboratories. Indeed, the method described in the American patent relates essentially to the analysis of systems in cell culture.
One of the objectives of the present invention is therefore to provide a new DNase method which makes it possible to carry out diagnoses on fixed tissues.
However, the subject of the present invention is furthermore, in general, methods intended to determine the state of a malignant cell in which the precise conformation of at least one gene within the nucleus of said cell is determined by labeling said gene in situ and evaluating the deviation of this conformation in relation to a normal cell as an index of its malignancy.
Indeed, while the determination of the position of the exposed DNA by the DNase method allows overall analysis of the state of malignancy of a tissue, on the other hand, the study of the precise conformation of at least one gene within a chromosome and the measurement of its deviation from the normal allows not only a classification of the various types of malignant cells, but also an evaluation of their malignancy index and of course, thereby, the monitoring of a therapeutic treatment.
It should be recalled that in the text which follows, the term “malignant” or “malignancy index” will also be optionally used to describe phenomena not directly linked to oncogenesis but also characterized by a disorganization of the chromosomes within the nucleus, for example cystic fibrosis.
This second method is based, here again, on the demonstration of the fact that by evaluating the conformation, that is to say both the position but also, if necessary, the structure of one or more genes by any appropriate technique, in particular by fluorescence in situ hybridization for example (FISH), it is observed that, depending on the malignant state of the cell, some genes involved in tumor suppression and/or activation move from the center of the nucleus outward and that their deviation from the normal constitutes a good sign of the state of the cell which is called hereinafter “malignancy index”, it being possible for this index to be evaluated by any appropriate method between a normal cell and a cell whose malignancy will be considered as irreversible.
This method allows a classification of tumors according to the position of the various genes in the nucleus and even of the three-dimensional structure of these specific genes in the nucleus.
In the first instance, it will be possible to study the position of the genes in the nucleus simply by studying the position of the centromers of a chromosome carrying them.
This method may be used with hybridization probes of the centromeric probe type; in this case, the normal corresponds to a peripheral location of the chromosomes carrying the centromeric probes and, in particular, as will be developed hereinafter, these centromeric probes may correspond to chromosomes 13q and 16q, which are carriers of HUMSIAH and Rbr-2 genes colocated on chromosome 16q and the Rb gene located on chromosome 13q, these genes being involved in the phenomenon of cancer suppression. It is understood that the centromeric probes which can be used in the methods according to the invention may correspond to any other cellular chromosome, in the same way that the gene labeling, according to the methods of the invention, may correspond to one or more genes carried on one or more chromosomes chosen from all of the chromosomes in the cell.
However, as has been indicated, the present invention also relates to a method intended to determine the overall state of malignancy of the cells of a tissue after fixing, characterized in that the exposed nuclear DNA is labeled and the entire DNA is labeled with a marker specific for the DNA, these two markers being capable of emitting uncontaminated specific signals.
“Uncontaminated specific signals” is understood to mean that it is possible to visualize separately, without notable contamination, the two signals corresponding to the two markers.
In general, the exposed nuclear DNA is labeled by the DNase I method, as was described above, and the DNA is labeled with the specific marker chromomycin A-3 (CA-3).
Among the exposed nuclear DNA markers, there should be mentioned more particularly tetramethylrhodamine used in combination with peroxidase. The labeling with peroxidase may be carried out by any appropriate method, in particular couplings of the avidin or streptovidin/biotin or DIG/antiDIG (DIG for digoxigenin) type.
Indeed, the peroxidase substrate, called tyramide-TRITC, makes it possible to amplify the signal obtained by a factor of 15 and thus makes it possible to analyze a fine distribution of the signal inside the nucleus.
The importance of using a marker specific for DNA is that it makes it possible to analyze the cell cycle in a semiautomatic manner, and therefore to make a correlation between the exposure of the genome obtained by means of the DNase I method and the cell cycle phases.
Thus, the present invention also relates to a method according to the invention, characterized in that the position of the exposed nuclear DNA relative to the cell cycle phase determined is evaluated by the marker specific for DNA as an index of the state of malignancy of the tissue cells.
In addition, the two fluorochromes thus used have an emission in the same region of the light spectrum, that is to say 600 nm, but their specific excitation is very different, 467 nm for CA-3 and 514 nm
Foley & Lardner
Forman B J
Zitomer Stephanie W.
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