WO2019135727A2 - Intervention with disorders of aging - Google Patents

Abstract

Determinants of organismal aging instrumental in occurrences of diseases of aging are described along with methods suitable for measurements of biological age and rate of aging of individuals in clinical laboratory practice.

Description

Intervention With Disorders Of Aging

BACKGROUND OF THE INVENTION

Investigations of populations of human and laboratory animals of varying ages show gradually decreasing capabilities of them in performances of various physiological functions with increase of age. Longitudinal studies of individuals in cohorts confirm the effects of aging on physiological functions and show that rates of declining with increase of calendar age can be significantly different from one individual to another depending on genotype and environment and lifestyle of individuals. Both cross-sectional and longitudinal investigations show increasing frequencies of particular diseases with increase of age. The diseases that are associated with aging have become leading causes of morbidity and death in most countries around world.

Attempts of treating diseased human antedate developments of the methods of pathology and laboratory medicine that improved accuracy of diagnosis and led to better understanding of how a particular disease occurs. Folk medicine practices of administering patients with substances collected from nature have been common and ancient in many societies despite borderline benefits or none of many. With developments in chemistry and pharmacology, purifications and making of particular molecules, e.g. from plants observed to cause an effect of interest in an animal species, have become possible to serve as a starting point to determine whether they can be used to develop a drug treatment beneficial in a particular disease in human. Many drug treatments practiced today are symptomatic treatments where a particular administration of a pharmaceutical formulation having a particular drug molecule has been shown to provide amelioration or disappearance of symptoms of a disease with tolerable unwanted effects in patients. Symptomatic treatments have been the usual treatment of diseases of aging. These are often chronic and administrations that alleviate persistent or recurring symptoms are common in the absence of a solution otherwise. Radical treatment of a disease differs from symptomatic treatments and typically requires correct understanding of its pathogenesis and it must be demonstrated that an identified upstream pathological event which must be distinguished from the typically multitudes of downstream pathological processes that also occur can be selectively intervened to produce cure without causing intolerable adverse effects in patients. Such treatments have been relatively infrequent in medical practice. The infectious disease treatments that target a particular molecular event that occurs in an infectious agent but not in the host (facilitating thereby selective intervention) are examples of such treatments.

Age at time of death and cause of death records of citizens available in several countries show that increase of the average lifespan of men and women during the last century has not been accompanied by a change in the maximum lifespan. Maximum lifespan of human is not found to show a significant difference in different times and countries where adequately large populations have been studied. Besides for human, maximum lifespan has been determined for various laboratory animal species and animals in wild and the results show that maximum lifespan is a genetically determined species- specific trait. A strong effect of genotype on maximum lifespan is emphasized by the large differences between species. For example, among mammals the common mouse has about 3 years of maximum lifespan and is burdened with several diseases of aging as it approaches 3 years of age whereas a 3 years old human is normally a healthy child.

Controlled experiments feasible with laboratory animals have shown a few environmental variables that could provide modest increases of maximum lifespan, such as restriction of the calories in diet. Even with the few means shown to cause about 10-20 % increase of maximum lifespan, across the board delays in occurrences of diseases of aging have been found. A few single gene modifications have also been found to cause modest increases of maximum lifespan in several species, usually proportionally more in species having naturally shorter maximum lifespan. Methods to make desired changes in nucleotide sequences of desired genes have been known and widely practiced in laboratory animals. Experience with these methods has shown that modifications and even complete deletions of some genes may be tolerated by animals but modifications of many genes turns out detrimental on health and lifespan of animals and the probability of a lethal outcome increases with simultaneous modifications of multiple genes.

Molecular genetic investigations of human populations around the world have shown common ancestors of them that populated the continents through stepwise migrations, settlements and mixings of populations. Whereas varying frequencies of particular gene alleles originating from population isolations and natural selection are observed around world (such as in genes affecting skin pigmentation that change with latitude), vast majority of the genes in human genome are found to be identical worldwide. Differences observed in the frequencies of diseases of aging in different countries are affected largely by environmental variables and by differences of age distributions in them.

Cancer is a disease of aging. Incidences of tumors of various organs are found to increase with increase of age in human and other species. Tumorigenesis is found to be a multi-step process requiring mutations and epigenetic modifications of several genes in a cell. Accumulations of mutations and epigenetic changes with increasing age and exposure to environmental mutagens are considered among the reasons of increase of tumors in older age groups and after such exposure. Patients having a tumoral disease are treated commonly by surgical excision of tumor where it is feasible. Where it is not feasible, one or more of the currently approved drug and/or radiation treatments are in general used. Objective therapeutic responses in tumor bearing men and women treated with the currently approved drug and radiation treatments of cancer have been assessed in numerous clinical investigations and in reviews of therapeutic data. The results show that for the majority of surgically incurable tumoral diseases, likelihood of cure is low and it is essentially zero in particular tumor subgroups. Long term follow ups of the patients treated by the conventional chemotherapy-radiotherapy of cancer show significantly increased occurrences of secondary tumors that are determined to be caused directly by them. Vast majority of the currently approved drug and radiation treatments of tumor bearing patients are known to act by causation of extensive damage to the genetic material of tumor cells and to cause irreversible genetic modifications in normal cells of patients.

DESCRIPTION OF THE INVENTION

This invention concerns effective interventions with aging and age-related diseases and also methods of determination of biological age and rate of aging of a person.

Great number and varieties of cells that interact locally and over long distances exist in human body and innumerable different molecules and reactions of them occur in each cell. How aging of a complex organism like human occurs and how the particular diseases associated with aging develop are accordingly prerequisites for effective interventions. In descriptions of my work about mechanisms of aging and pathogenesis of particular age- related diseases, I have pointed that whereas aging of an organism is affected by supracellular interactions, aging of organism is driven primarily by the cellular aging deriving from intrinsic failures of cells (Ta§ S, Monographs In Developmental Biology, 1984;17:178-192). It was pointed in that respect that aging is not a universal feature of all life forms. Prokaryotic organisms can be clonally propagated without limit or signs of aging whereas even the unicellular eukaryotes show aging and limited clonal lifespan. Aging is a characteristic of eukaryotes. The unicellular eukaryotes avoid the extinction that would be caused by the limitation of clonal lifespan by undergoing meiosis that provides rejuvenation similar to the meiotic rejuvenation in higher eukaryotes. In human and other multicellular eukaryotes somatic cells show aging but germ line cells can give rise to youthful progeny even at advanced ages that exceed the average lifespan of species. Further relevant to aging are the findings that whereas normal somatic cells taken from various tissues show limited lifespan in vitro and also in vivo when serially transplanted in inbred animals that do not show histoincompatibility with them, neoplastic cells typically show unlimited lifespan under the same conditions. Normal somatic cells reaching the end of clonal lifespan show several features of the normal somatic cells seen in tissues of older subjects and the numbers of cell divisions occurring before the end of clonal lifespan are found to correlate with species-specific maximum lifespan potential in several species.

Eukaryotic organisms differ from prokaryotes in structure of genetic material and usually also in having mitochondria that allow generation of far greater number of ATP per glucose molecule or equivalent by oxidative metabolism than that available by nonoxidative metabolism. Eukaryotic DNA is extensively complexed with particular proteins to make the chromatin complex wherein accessibility of DNA is highly restricted unlike the situation in prokaryotes. Analyzing the common denominators of the life forms that do not show aging, the molecular events pertinent in meiotic rejuvenation and my experimental findings about aging, I have pointed to the particular modifications of the structure of eukaryotic genetic material during cellular differentiation and with increase of age of organism as being instrumental in the occurrence of biological aging (Ta§ S, 1984).

Wrapping of eukaryotic DNA around nucleosomal core histones, folding of polynucleosomal fiber to form the 30 nm wide chromatin fiber, side-by-side packing of such and formations of loops of chromatin fiber contribute to the packing of over a meter of genomic DNA (e.g. in human) into a nucleus that typically has a diameter less than 1/10 000 000 of the total length of genomic DNA. The restricted access to DNA and the regulation of access that are made possible by the structure of chromatin allow differential patterns of expressions of the genes of genome in a stable and inheritable manner and allow thereby formations of cells of very different phenotypes despite having the same genotype. The cellular differentiation made possible in this manner is key to the development of advanced multicellular eukaryotes but has its costs in the occurrence of aging. Repression of particular genes and regions of genome by the packing of DNA in a condensed chromatin structure (heterochromatin), necessary for cellular differentiation, creates problems, particularly in the repair of damage to the genetic material as evidenced by the less efficient repair of DNA damage in heterochromatin than in euchromatin, and the problem is compounded by the oxidative metabolism-generated oxidants that form an internal source of threat to the integrity of genome besides the external threats, e.g. by mutagens in environment ( Ta§ S, 1984). For example alkali-labile DNA lesions (apurinic- apyrimidinic sites that can be caused by oxidants) and strand breaks show significant increases with increasing age of mice in liver cells and in other tissues (Ta§ S et al, Gerontologist , Part II, 1978; 18: 131). Also the facultative heterochromatinization of one of the X chromosomes in female mammals early during development with differentiation of embryonal stem cells is a known example of the differentiation associated condensation of chromatin and the greater increase of mutations with increasing age in the inactivated X than in the active X in the same cells of aging women (e.g. Machiela MJ et al, Nature Communications 2016;7: 11843) confirms the limitations posed to the maintenance of the integrity of genome by its modifications that are necessary for cellular differentiation.

The chromatin complex as it exists in cells is challenging for elucidation of structure by the methods commonly used for macromolecular complexes. It is many magnitudes greater in size and number of components relative to the largest macromolecular complexes whose structures have been determined. Various methods have accordingly been developed to tackle the structure-function relationships in chromatin. In my work into the structure of chromatin and its changes during cellular differentiation and aging, 1 used an approach of studying both the intact complex and step-wise dissociations of its constituents. Experiments where live normal cells are rapidly lysed on top of neutral sucrose gradients at 2-4 °C by a nonionic detergent in hypotonic or isotonic medium followed by isokinetic and equilibrium density gradient centrifugation analyses of the released demembranized nuclei with and without reduction of the disulfide (S-S) bonds existing in them have revealed that aging of organism is associated with significant modifications of structure of chromatin in various tissues. Comparisons of rates of sedimentation of demembranized mononuclear spleen cell nuclei between immature, young adult and old mice have shown significantly increased rate of sedimentation with increasing age of animals with nuclei in which the S-S bonds were kept intact whereas an opposite influence of the animal age on sedimentation rates was found upon reduction of the S-S bonds in the nuclei which caused marked decondensation of chromatin with nuclei of old mice and relatively less decondensation with nuclei of younger mice (Ta§ S et al, Mechanisms of Ageing and Development 1982;19:73-84). A similarly greater

decondensation of chromatin of the liver cells of old than young adult mice upon reduction of the S-S bonds in nuclei was also found (Ta§ S et al, Mechanisms of Ageing and Development 1980;12:65-80). Control experiments where cells of immature and old mice were mixed prior to lysis together have shown that the demebranized nuclei continue to show the age-specific sedimentation rates of the unmixed cells of respective age groups, that the decrease of rate of sedimentation upon treatment of nuclei with S-S reducing agents is due to decondensation of them as verified also by microscopic visualization, and that the demembranized nuclei of old mice sediment slower than even those of immature mice upon reduction of the S-S bonds in nuclei in sharp contrast to the opposite when the S-S bonds existing in the nuclei are kept intact (Ta§ S et al, 1982). Thus an increased S-S mediated condensation of chromatin complex was found with increasing age of animals that coexisted with a decondensation of structure of chromatin revealed upon reduction of the S-S bonds in the nuclei.

The decondensation of chromatin in cells of various tissues with increasing age of animals revealed upon reduction of the S-S bonds in nuclei has been confirmed by different methods as well. Kinetics of endonucleolytic cuts to DNA in chromatin in liver cell nuclei of mice of varying ages have shown that a fraction of chromatin having less than 10 % of nuclear DNA gets preferential cuts when the nuclei are incubated briefly with limited concentrations of deoxyribonuclease I (DNAse 1) or micrococcal nuclease and this fraction, soluble in 3-5 mM Mg⁺⁺, makes a lower proportion of total chromatin in cells of old than young adult mice whereas a greater difference exists in the opposite direction between young adult and old mice with respect to another fraction of chromatin that makes about 70-75 % of total chromatin under the same conditions of cuts to nuclear DNA [Ta_§ S et al, 1980; Ta§ S et al, Journal of Gerontology 1982;37:673-679

(abbreviated Ta_§ S et al, 1982b)]. This second fraction of chromatin is solubilized in hypotonic buffer that lacks divalent cations. It is enriched for constitutive heterochromatin as shown by its content of ~ 70 % or more of the nuclear DNA and was found to get more endonucleolytic cuts in liver cell nuclei of old than young adult mice in incubations with limited concentrations of DNAse I or micrococcal nuclease or endogenous endonucleases alone, pointing to occurrence of a more open conformation of it with increasing age (Ta§ S et al, 1980; l982b).

Equilibrium density gradient analyses of the regions of chromatin that remain bound to nuclear lamina-matrix of liver cell nuclei following removal of the endonucleolytically cut fragments of chromatin have shown significantly greater density when obtained from liver cell nuclei of old than young adult mice with nuclei in their native state with intact S-S bonds and shown near equalization of density upon treatment of them with S-S reducing agents (Ta§ S et al, 1980). In accord, a separate analytical method has shown that intact S-S bonds of a subpopulation of tightly DNA bound nonhistone proteins are instrumental in linking different sites of nuclear DNA with each other in normal mononuclear spleen cells and reduction of these S-S bonds causes marked unfolding of the intact supercoiled nuclear DNA that is looped and anchored at nuclear lamina-matrix (Tas S et al, 1982). The intact nuclear DNA and tightly bound nonhistone proteins that are resistant to dissociation by high ionic strength (2.0 M NaCl) have been purified by ultracentrifugal separation by taking advantage of the supercoiling of nuclear DNA when it is effectively circularized by looping of it by DNA bound proteins. The nuclear DNA-tightly bound nonhistone protein complexes purified as above were then incubated with restriction endonucleases to generate restriction fragments of the nuclear DNA having tightly bound nonhistone proteins at particular sites and were analyzed by gel electrophoresis with and without treatment of them with S-S reducing agents. These analyses have further revealed that particular sites of nuclear DNA separated from each other by tens of kilobase pairs or more along the linear genomic sequence are physically linked to each other by tightly DNA bound nonhistone proteins whose S-S bonds are necessary for keeping these DNA sites linked. Evidence of the looping of the nuclear DNA by tightly bound nonhistone proteins to form loops predominantly in the 60-110 kb loop size range has also been found (Ta§ S et al , FEBS Letters 1985;191 : 136-140). Cells of spontaneously occurring tumors of old mice and corresponding normal tissue cells of tumor-free age-matched and young adult mice have been compared with respect to the structure of chromatin for insights into the mechanisms of aging and of tumorigenesis that accompanies aging. Intact nuclear DNA with tightly bound nonhistone proteins resistant to dissociation by 1.6-2.0 M NaCl were purified from spontaneous lymphoma cells and from normal lymphocytes and analyzed as described in Ta§ S et al, 1982 and by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). These investigations have shown that chromatin of spontaneous tumors of old mice has features similar to the normal cells of young mice rather than those of normal cells of old mice with regard to the redox state of DNA associated proteins and revealed a protein having 213 ± 24 kD (mean ± S.D.) apparent molecular mass by SDS-PAGE as a major constituent of the intact nuclear DNA- tightly bound protein complexes [Ta§ S et al, Age l980;3:95 (abbreviated Tas S et al,

1980b)]. Little or no detectable entry of the 213 ± 24 kD protein into gels was seen in SDS-PAGE analyses of intact supercoiled nuclear DNA-protein complexes of normal lymphocytes of old mice under nonreducing conditions whereas reduction of the S-S bonds in these complexes prior to loading to gels caused greatly enhanced entry of the 213 ± 24 kD protein into gels along with certain higher molecular mass proteins. The increase of entry of the 213 ± 24 kD protein into gels upon reduction of S-S bonds was found to be less with the complexes of young adult mice and least with those of lymphoma cells of old mice, pointing to modifications of it during aging and targeting of the protein during neoplastic transformation (Ta§ S et al, 1980b).

Methods to analyze decondensation of chromatin by disulfide reducing agents in individual cells have been developed in view of heterogeneity of cells in normal tissues and tumors. Normal live cells of human and their neoplastic counterparts were lysed at ~ 2 °C by a nonionic detergent in buffers having physiological or increased salt concentrations and a blocker of sulfhydryl groups against artefactual oxidation and sulfhydryl-disulfide exchange reactions of proteins upon lysis of cell membranes and the individual demembranized nuclei of these cells were examined by microscopy and by additional methods with and without reduction of the disulfide bonds existing in the nuclei. With freshly obtained nuclei prepared at physiological ionic strength, reduction of the S-S bonds existing in the nuclei of cells has been determined to cause decondensation of chromatin of every normal cell of the peripheral blood mononuclear blood cells of healthy human [Ta_§

S et al, Cytologia 1985;50:405-415 (abbreviated Ta§ S et al, 1985b)]. Examinations of acute and chronic lymphocytic leukemia (ALL and CLL, respectively) cells of nontreated patients in the same investigations have shown significantly smaller or no detectable decondensation of chromatin by S-S reducing agents in neoplastic cells. Mixing of normal and neoplastic cells at known proportions before lysis together verified that the determined differences of structure of chromatin of normal and neoplastic cells existed in situ in the live cells and could not be due to potential action of cytoplasmic factors on

demembranized nuclei during the brief interval they were exposed to cytosol (Ta§ S et al,

1985b). These experimental results in comparisons of neoplastic and normal cells in human accorded with the aforementioned findings on structure of chromatin in spontaneous lymphoma cells and normal spleen cells of old mice.

Causation of step-wise dissociations of the DNA bound molecules from the intact nuclear DNA in situ in demembranized cell nuclei by increasing the ionic strength of medium has shown causation of practical invisibility of them under light microscope around and above the ionic strength of 0.6 M NaCl, i.e. upon causation of dissociation of histone HI from DNA, and shown revisualization of them by inclusion of adequate concentrations of a positive supercoiler of DNA (ethidium bromide) in the medium. Since ethidium bromide can cause positive supercoiling of DNA when it is circular or effectively circularized by the looping of DNA, these findings have shown further evidence of the loops of chromatin normally in cell nucleus and shown dependence of the packing of polynucleosomal fiber within the confines of nucleus on histone Hl and other proteins dissociated from DNA by 0.6 M NaCl. They evidenced also the anchoring of the loops of chromatin fiber with elements of a nuclear protein skeletal structure existing at 0.6 M NaCl ionic strength. Exposing these complexes of nuclear protein skeletal structures (nuclear lamina-matrix) and attached loops of chromatin fiber to S-S reducing agents at 0.6 M NaCl has shown causation of marked decondensation of the complexes when obtained from normal cells of human and has shown consistently smaller decondensation when obtained from the neoplastic counterparts of normal cells (Ta_§ S et al, l985b). Irradiation of normal lymphocytes on ice with varying doses of g-rays to cause varying numbers of DNA strand breaks, followed by immediate preparations of the complexes of nuclear lamina-matrix-polynucleosomal fiber loops as above confirmed that the S-S reducing agents-induced decondensation and increase of the diameter of the complexes occurs through reduction of S-S bonds existing at the bases of loops so as to convert them to longer loops which could not occur when the DNA of a loop was broken (Ta_§ S et al, l985b). The lowest g-irradiation dose at which the S-S reducing agents induced decondensation of these complexes became insignificant (400 rads) had been determined as the LD50 of single whole body irradiation of human.

Intact supercoiled nuclear DNA-lamina-matrix complexes of normal mononuclear cells and of leukemia cells of human subjects were purified by ultracentrifugal separation from the rest of cellular constituents to identify the molecular components whose change of redox state, structure, DNA binding or abundances are behind the determined consistent differences of the structure of chromatin between the normal and neoplastic cells. A silver staining method that provides simultaneous visualizations of the protein and nucleic acid components of the complexes in gels following their electrophoretic separations was used. Such SDS-PAGE analyses have revealed multiple intermolecular S-S bonded proteins in the complexes prepared from normal mononuclear blood cells and shown lack of S-S bonding of several but not all of the proteins in leukemia cells (Ta§ S, 1984). A protein showing apparent molecular mass of ~ 220 kD by SDS-PAGE has been found as a major protein species of the complexes of normal mononuclear blood cells of human and shown marked increase of entry to gels upon treatment of the samples with S-S reducing agents prior to loading to gels similar to the likewise identified 213 ± 24 kD protein of the complexes of normal splenic mononuclear cells of mice (Ta§ S et al, 1980) and amount and S-S bonding of this protein showed marked decreases in the complexes of leukemia cells in comparison to those of normal cells (Ta§ S, 1984).

In nonreducing SDS-PAGE analyses of purified intact nuclear DNA-tightly bound protein complexes wherein the complexes were not treated with DNAse 1 prior to denaturation of samples by 1.2 % SDS at 100 °C for 10 minutes and had accordingly variously sized single stranded DNA molecules broken by such denaturation and by pipetting of samples, the above mentioned ~ 220 kD protein has shown a diffuse band unlike the sharp bands of other proteins in the gels (Ta§ S, 1984). A smear of silver stained material was seen immediately below the ~ 220 kD protein in the gels and continued downwards with decreasing intensity. Such smears were not seen with samples digested with DNAse I prior to loading to gels. Reduction of the S-S bonds in the samples of complexes that were not subjected to DNAse I digestion prior to loading to gels has shown simultaneous causations of

(1) sharpening of the ~ 220 kD protein band, (2) disappearance of the smeared silver staining below the ~ 220 kD protein band, and

(3) markedly increased quantity of the ~ 220 kD protein detected in gels with complexes of normal mononuclear blood cells in comparison to the nonreduced samples of the complexes of same cells (Ta§ S, 1984). In addition SDS-PAGE of DNA and protein molecular size markers and fractions of chromatin side by side in slab gels have shown that linear double stranded DNA molecules of ~ 2 kb migrate around the position of the

~ 220 kD protein in gels and that SDS-PAGE provides increased electrophoretic mobilities of DNA molecules compared to the conventional PAGE of DNA so that in the presence of SDS < 2 kb double and single stranded DNA molecules elute off the gels under the SDS- PAGE conditions used in the investigations described in Ta§ S, 1984 (Ta§ S, Analytical Biochemistry 1990;188:33-37). The above described investigations have thus revealed that particular nonhistone protein molecules that are not dissociable from nuclear DNA of normal tissue cells by high ionic strength (2.0 M NaCl) include a protein species having apparent molecular mass of ~ 220 kD and it is resistant also to dissociation by 1.2 % SDS and that treatment of these complexes with S-S reducing agents causes dissociation of DNA from the ~ 220 kD protein. The identified ~ 220 kD protein species has been found to show age-associated increase of abundance in the intact nuclear DNA-lamina-matrix complexes prepared from normal tissue cells of human and mice (Ta§ S, 1984).

Aforementioned alterations of chromatin structure determined to occur in cells in various normal tissues with the increase of age of organism have the evidence of being instrumental in declining of normal tissue-organ functions during aging and also in occurrences of diseases of aging. Methods of detection of them that are suitable for clinical laboratory practice would accordingly be useful in objectively determining and monitoring rate of aging of individual human subjects and in assessments of preventive and therapeutic interventions. Yet chromatin is a very large macromolecular complex and changes of structure of chromatin during aging are also complex and include changes in opposite direction at different levels of chromatin architecture.

Blood specimens are routinely obtained from human subjects for various clinical laboratory tests. Some tests are performed with blood plasma or serum and others with blood cells. Unused portions of blood specimens are ordinarily discarded after the tests. I carried out investigations with mononuclear blood cells of a selected subset of these blood specimens with regard to the alterations of chromatin during aging by selecting specimens whose analyses showed no evidence of disease and where unused amount of an anticoagulated specimen had adequate number and viability of the mononuclear blood cells at the start of chromatin structure analyses in the afternoon of blood collection.

Ethical clearance for these investigational uses rather than discarding of unused portions was received in the hospitals where I served as consultant of clinical laboratories.

Mononuclear blood cells were obtained from each specimen by standard methods and their viability was determined by Trypan Blue dye exclusion. Cells of specimens showing > 90 % viability were lysed with a buffer having a nonionic detergent (Nonidet-40; NP-40) to prepare nuclei of cells as described below in separate tubes for each specimen placed into crushed ice. Nuclei from subjects in the age groups 18-29 years (young) and > 50 years (older) were pooled in two tubes for the respective age groups, brought to equal concentration by measurements of A260 (absorbance at 260 nm) and the chromatin fractionations described below were carried out in parallel with equal numbers of demembranized nuclei of each age group.

Live cells in phosphate buffered saline (PBS) were lysed by addition of 20 fold or greater volume of ice-cold cell lysis buffer having 1.2 % NP-40 in 0.32 M sucrose, 10 mM NaCl, 3 mM MgCl₂, 5 mM ethylene glycol-bis^-aminoethyl ether)-N, N, N’, N’- tetraacetic acid (EGTA), 2 mM N-ethylmaleimide (NEM), 5 mM iodoacetic acid (IAA), 0.2 mM phenymethylsulfonyfluoride (PMSF), 10 mM Tris pH 7.4. Features of a suitable cell lysis buffer include near isotonicity, adequate inhibitors of sulfhydryl oxidation and of sulfhydryl-disulfide exchange reactions, of Ca⁺⁺ triggered reactions and of proteolytic reactions that can occur upon lysis of cellular membranes and adequate [Mg⁺⁺] to maintain conformation of chromatin like in the cells prior to lysis. Brief duration in the assays from the lysis of live cells to the obtaining of demembranized nuclei and the low temperature during lysis also facilitate preservation of structure of chromatin like in the cells prior to lysis. Control experiments where (1) cells of young and older age subjects were mixed immediately before lysing together and (2) cell nuclei of young subjects were exposed to cytosol of older subjects, and vice versa, have shown no detectable effect of cytosol on the donor age associated changes of structure of chromatin determined as described here.

Tubes having live cells in PBS were placed into crushed ice, the cell lysis buffer was added onto the cell suspension as described above and tubes were inverted a few times for mixing. Within approximately 5 minutes of the addition of cell lysis buffer, tubes were centrifuged at 450 g for 10 minutes at 2 °C. The supernatant (cytosol) was discarded unless to be assayed in other tests. The pelleted nuclei were resuspended in the cell lysis buffer lacking NP-40. Suspensions of demembranized nuclei were adjusted to equal concentration in experiments comparing different groups (e.g. young vs older subjects, normal vs neoplastic cells) and 2.2 ml aliquots were pipetted to polypropylene

microcentrifuge tubes placed into crushed ice. Nuclei concentrations above A260 of 1.0 could be used for the chromatin fractionations described here to obtain acceptable precision in duplicates in the light scattering determinations of fractions in 2.2 ml volumes. Higher concentrations up to about A260 = 6.0 of the nuclei could be used advantageously but require greater number of starting cells. Measurements of the light scattering of particles with instruments designed for measurements with ten fold or smaller volumes can provide improvements of precision and accuracy in assays with relatively small number of cells and are advantageous in automation of assays.

Tubes having aliquots of demembranized nuclei were placed into crushed ice and divided to two subgroups where one received a disulfide reducing agent (commonly 2- mercaptoethanol) to 45 mM final concentration and the other half received 7 mΐ ethanol. Other suitable disulfide reducing agents, e.g. a-thioglycerol, can also be used at a final concentration that provides molar excess over the disulfide bonds of proteins and over other sulfhydryl reactive molecules in samples. After inversion a few times, the tubes received DNAse I at the final concentrations described below in results. Final DNAse 1 concentrations were evaluated in the 10-100 Units/ml range in pilot tests along with the absence of it for assessments of the endonucleolytic cuts to DNA by endogenous endonucleases alone. Tubes were incubated in crushed ice (~ 2 °C) or at room temperature (~ 23 °C) or at 37 °C for periods up to 30 minutes as indicated in the descriptions of results. Ethylenediaminetetraacetic acid (EDTA) was added to tubes at the end of tested periods at 4 mM final concentration and tubes were centrifuged at 2300 g for 7 minutes at 2 °C. The supernatants (I^st supernatant fraction, 1S) were collected and 2.2 ml of a hypotonic buffer containing 0.5 mM Mg⁺⁺ (0.5 mM MgCl₂, 0.2 mM EGTA, 0.2 mM PMSF, 10 mM Tris pH 7.4) was added onto pellets. Tubes were vortexed for about 15 seconds, placed into crushed ice and were centrifuged after approximately 5 minutes in ice at 2300 g for 7 minutes at 2 °C to separate the soluble chromatin fragments released by the hypotonic lysis from the residual nucleus. The supernatant (2^nd supernatant fraction, 2S) were collected and 2.2 ml of 2.0 M NaCl, 0.5 mM MgCl₂, 0.2 mM EGTA, 0.2 mM PMSF, 10 mM Tris pH 7.4 was added onto pellets. Tubes were vortexed for about 15 seconds, placed into crushed ice and were centrifuged after about 5 minutes at 2300 g for 7 minutes at 2 °C to separate the DNA fragments dissociated by 2.0 M NaCl from the final residual nucleus (nuclear lamina-matrix with the attached regions of nuclear DNA undissociable by 2.0 M NaCl). The supernatants of this last centrifugation (3^rd supernatant fraction, 3S) were collected and 2.2 ml of the same 2.0 M NaCl containing buffer was added onto pellets. The pelleted residual nuclear material (nuclear lamina-matrix with the attached regions of nuclear DNA undissociable by 2.0 M NaCl, P) was suspended by vortexing for about 15 seconds. The above described method of nuclear fractionation and analysis of structure of chromatin provides information about the accessibility of DNA in chromatin in situ and also about the three dimensional organization or architecture of genetic material within the nuclear space. The individual steps producing fractions 1S, 2S, 3S and P allow determinations of molecular components in each (including the genomic sequences) besides providing information about three dimensional organization of genetic material in cell nucleus as described here. The assay conditions, including the buffer compositions and endonuclease, are exemplary and can be varied as long as fractions are produced that reflect the structure of the chromatin complex existing in live cells. The endonuclease added into the incubation medium of nuclei in the examples (DNAse I) has a molecular mass of about 31 kD and can introduce cuts to the accessible DNA essentially independent of nucleotide sequence. Besides other such endonucleases, suitable restriction

endonucleases are also available that can be used to introduce cuts to the DNA in chromatin depending on both the accessibility and nucleotide sequence of DNA. The 1 S, 2S, 3S and P obtained as above were analyzed as described below.

Size and conformation of double stranded DNA molecules and of DNA-protein complexes can be determined by various methods with accuracy and precision that depend on the method and subject molecules and complexes. Testing has shown that centrifugal separation through neutral sucrose density gradients can be used for detecting the donor age associated changes of conformation of large particles such as the demebranized whole nuclei in isotonic and hypotonic buffers but resolution and precision of such methods become unsatisfactory in detection of differences of chromatin fiber length in

fractionations similar to that described above except for the step separating 3S and P (Ta§ S et al, 1980, 1982). Electrophoretic separations of DNA molecules can provide determinations of length with single nucleotide resolution within a relatively narrow size range and can be informative for molecules over broad size ranges but show lowering of power for very large DNA molecules such as those that occur in chromosomes and subdomains thereof. Density gradient and electrophoretic separation analyses are also relatively time consuming to lower suitability for clinical laboratory practice.

Turbidimetric-nephelometric methods are based on determination of the scattering of light passing through a solution or suspension. Particles in solution or suspension cause scattering of light in proportion to their size and concentration. Wavelength of light also affects the signal detected by a photodetector which is typically arranged at zero degree relative to the path of incident light in turbidimetric measurements and at 90° in conventional nephelometry. Lower angle arrangements of photodetector can be used and provide advantages. Optimal wavelength for a turbidimetric or nephelometric method depends on the size range of analyzed particles and can be determined by known methods. In general lower wavelengths can provide greater signal but there are limits to its lowering due, e.g. to occurrences of absorbances by particular molecular species in the particles. Evaluation of wavelengths for the light scattering measurements described here has shown that wavelengths in the 320-600 nm range can be used with the particles generated by varying numbers of endonucleolytic cuts to DNA in nuclei. Measurements at 380 nm provided satisfactory precision and sensitivity in detection of the donor age associated changes of chromatin structure in the examples described here. In general larger particles show greater settlement from suspension with time. Addition of viscosity increasing agents to the solutions or suspensions that are assessed by measurements of light scattering can be used against errors of measurement arising from settlement of particles. Such agents were not necessary for the light scattering measurements described here for 1S, 2S, 3S and P performed with 10 mm path length quartz cuvettes with a spectrophotometer capable of multiple measurements at desired wavelengths repeatedly. Effects of settlements of particles during a period of measurement can be determined with such instruments by repeats of measurements at desired intervals. Inverting stoppered cuvettes a few times immediately prior to the light scattering measurements with 1S, 2S, 3S and P was found to be adequate for avoiding errors from settlement. ephelometry, particularly in combination with a flow cell, provides advantages in automation of the methods of analysis of structure of chromatin that are described here for use in clinical laboratories.

The fractionation of chromatin described here has advantages. The process starting with live cells until the demembranized nuclei are obtained is brief, at cold (around 2-4 °C) and the demembranized cell nuclei are kept in a buffer that maintains

conformation of chromatin like in live cells. In such nuclei the endonucleolytic cuts to DNA in chromatin by DNAse 1 or by a similar endonuclease is limited essentially by the accessibility of DNA to the externally added endonuclease and to endogenous

endonucleases. A chromatin fragment produced by cuts to double stranded DNA is released from the remainder of demebranized nucleus if the fragment is not tethered thereto by a chromatin protein and the cut fragment is solubilized if the buffer in which the nucleus exists is permissive to its solubility. The buffer with 3 mM Mg⁺⁺ in which the nuclei are suspended during the endonucleolytic cuts to DNA and into which the 1 S fragments are released is permissive particularly to solubility of chromatin fragments that are enriched in hyperacetylated histones that help to provide a relatively unfolded, open chromatin conformation. The hypotonic buffer in which the fragments of 2S are soluble is conducive to solubility of most of cut fragments that are not tethered to the remainder of nucleus by a chromatin protein. The buffer with 2.0 M NaCl in which the fragments of 3S are soluble can solubilize a cut fragment unless it is tethered by a tightly DNA bound nonhistone protein that resists dissociation by 2.0 M NaCl and continues to be bound to an element of the nuclear lamina-matrix by bonds resistant to 2.0 M NaCl. Histones and most nonhistone proteins are dissociated from nuclear DNA at the high ionic strength of 2.0 M NaCl to leave behind free DNA fragments and DNA fragments bound at particular sites by proteins undissociable by 2.0 M NaCl. The final pellet (P) contains the nuclear lamina- matrix with the attached regions of DNA that are not dissociated by 2.0 M NaCl.

Kinetics of the endonucleolytic cuts to DNA and productions of 1S, 2S, 3S and P were examined by incubations of demembranized nuclei with 0-100 Units/ml DNAse I for 0-30 minutes at ~ 2 °C or ~ 23 °C or 37 °C with and without reduction of the disulfide bonds existing in the nuclei. With demembranized nuclei of normal human mononuclear blood cells with intact disulfide bonds, about 25-30 % of total nuclear DNA remained at nuclear lamina-matrix (P) when the nuclei prepared as above were incubated for 0-10 minutes at ~ 2 °C and incubation at ~ 23 °C caused about 20 % of the DNA to remain at P. On the other hand, lysis of live cells at ~ 2 °C with a nonionic detergent in the presence of 2.0 M NaCl and EDTA for blockade of the divalent cations required by endogenous endonucleases followed by separation of the nuclear lamina-matrix-DNA from the rest of cell constituents has shown that essentially 100 % of the nuclear DNA remains bound at nuclear lamina-matrix under such conditions. Thus even the relatively few endonucleolytic cuts to the DNA in chromatin in situ during preparation of nuclei at ~ 2 °C suffice to cause release of more than half of nuclear DNA from lamina-matrix when such nuclei are fractionated as described here. Further, addition of DNAse I to the incubation medium of demembranized nuclei has shown causation of rapid decrease of the DNA remaining at lamina-matrix to ~ 10 % or less of total nuclear DNA within only a few minutes at ~ 23 °C and then only a few percent further decrease of it with the increase of incubation up to 30 minutes with 50 Units/ml DNAse I. Still further, increase of the DNAse I concentration up to two fold had relatively little effect in this respect. All of these results accord with the folding of nuclear DNA-chromatin fiber into loops and attachments of such at relatively few sites to nuclear lamina-matrix.

Kinetics of the formation of 2S fragments that are released from the remainder of nucleus by hypotonic lysis have also shown that in comparison to the absence of added DNAse I, addition of 50 Units/ml DNAse I to the incubation medium of nuclei caused a sharp (up to three fold) increase of the nuclear DNA released from the remainder of nucleus into 2S within only a few minutes of addition of DNAse I at ~ 2 °C or ~ 23 °C and relatively little change afterwards with incubation up to 30 minutes. These results with normal human mononuclear blood cell nuclei are similar to those with the nuclei of normal tissue cells of mice (Ta§ S et al, 1980, 1982b) and are also consistent with the folding of nuclear DNA into loops and attachments of loops to elements of nuclear lamina-matrix in such a manner that relatively small number of DNAse I cuts to the DNA in chromatin in a few minutes suffice for release of over 70 % of nuclear DNA into (1 S + 2S) and over 90 % into (IS + 2S + 3S) from the lamina-matrix while magnitudes longer exposure to identical DNAse I concentrations afterwards causes little further release of DNA therefrom. Similar kinetics of the release of nuclear DNA into (IS + 2S) and into (1 S + 2S + 3S) and the production of (1S + 2S) without exposure of the nuclei to non-physiological high ionic strength rule out potential precipitations of nuclear proteins by 2.0 M NaCl to form a nuclear protein skeleton (lamina-matrix) to which loops of chromatin fiber are attached.

Investigations of normal mononuclear blood cells of human subjects by the methods described above have shown that age of the subject is a significant determinant of structure of chromatin in normal cells. Analyses of 2S and 3S fractions by measurements of light scattering and the normalizations of light scattering for concentration (with A260 for DNA as well as with A280 for protein) indicated significantly decreased average size of the particles of 2S and 3S with increase of donor age (Table 1). Decrease of the average particle sizes in the 2S and 3S fractions with increase of donor age was confirmed also by gel electrophoretic analyses of DNA of 2S and 3S fractions. Statistically significant effect of donor age on structure of chromatin was detectable with nuclei in which the

endonucleolytic cuts to DNA in chromatin were made by endogenous endonucleases alone as well as when they were made also by externally added endonuclease DNAse I. Both light scattering and electrophoretic analyses have shown that far more endonucleolytic cuts are made to the DNA in chromatin in situ by as low as 10 Units/ml DNAse I than by endogenous endonucleases alone (e.g. at ~ 2 °C in 5 minutes). The difference between young and older subjects in terms of the average 2S and 3S particle sizes become smaller with increase of concentration of DNAse I in the medium of nuclei and with prolongation of incubation although statistically significant difference between the young and older age groups persists with DNAse 1 concentrations increased as much as to 50 Units/ml DNAse I with incubations for 5-30 minutes. It would therefore be preferable to use relatively low concentrations of the added endonuclease and a brief incubation period that allow' detection of the effect of aging on the accessibility of DNA in chromatin in uses of the method in clinical laboratory practice in assessments of biological age of human subjects.

The 2S and 3S fractions in which the average particle sizes decrease with increasing age of men and women constitute large proportions of the total nuclear DNA. The 2S contains over a third and (2S + 3S) contains more than half of genomic DNA already after a few minutes of endonucleolytic cuts to DNA in chromatin by DNAse I, indicating that 2S and 3S particles are generated predominantly from constitutive heterochromatin. The remarkable degree of the influence of donor age on the structure of chromatin shown in Table I points thus to significant increases in the accessibility of DNA in heterochromatin in normal tissue cells in human. This particular effect of the aging of organism on structure of chromatin in normal mononuclear blood cells was observed regardless of the treatment of nuclei with disulfide reducing agents and is similar to the findings in normal liver cells and mononuclear spleen cells of aging mice analyzed by time consuming methods not suitable for clinical laboratories (Ta§ S et al, 1980, 1982, l982b).

The nuclear lamina-matrix-DNA complexes (fraction P) has shown causation of marked decrease of the light scattering by them within a few minutes of the incubation of nuclei with DNAse I in association with a sharp decrease in the proportion of the nuclear DNA remaining bound at the complexes, indicating that the DNA molecules remaining bound at lamina-matrix contribute to the light scattering by the complexes. Table II shows that the particles of P have shown significantly smaller light scattering when produced from mononuclear blood cells of the men and women in the > 50 years age group than in the 18-29 years age group. These test results accord with those in Table I that indicate that the DNA in constitutive heterochromatin shows increase of accessibility to endonucleases so as to undergo more cuts per unit time when the nuclei are from cells of older persons.

The nuclear lamina-matrix-DNA complexes of normal mononuclear blood cells have shown significant decreases of light scattering also when the nuclei from which they were obtained were treated with disulfide reducing agents (Table II). Disulfide reducing agents have shown this effect both with complexes from nuclei in which the endonucleolytic cuts were made by endogenous nucleases alone and also from those cut by up 100 Units/ml DNAse I at ~ 2 °C or ~ 23 °C or 37 °C. Decrease of light scattering of the particles of P upon reduction of the disulfide bonds in them is accompanied by causation of release of DNA from them when they are produced from normal cells. The proportion of the nuclear DNA released from P upon treatment of nuclei with disulfide reducing agents is relatively more when the nuclei are incubated with lower concentrations of DNAse I. The 3S obtained from the same nuclei with and without reduction of the S-S bonds of the proteins bound to the 3S DNA has also shown significantly decreased light scattering of the 3S particles upon reduction of the S-S bonds. The DNA molecules in 3S, like those in P, have been exposed to 2.0 M NaCl and accordingly are mostly bare DNA except for the sites that are tightly bound by particular nonhistone proteins that are undissociable by 2.0 M NaCl. The 3S DNA is released from the lamina-matrix by 2.0 M NaCl and is soluble in it whereas the DNA of P remains tethered thereto.

The disulfide reducing agents induced decrease of the light scattering of the particles of 3S and of P accord with the results of gel electrophoretic analyses of their DNA and protein components under disulfide reducing and nonreducing conditions. Simultaneous visualization and analyses of the DNA and protein components of 3S and of P by SDS- PAGE as described in Ta§ S, 1990 have shown significantly increased electrophoretic mobilities of the DNA molecules of them upon reduction of the S-S bonds in them similar to the results described in Ta§ S, 1985. Since SDS-PAGE analyses of purified restriction fragments of bacteriophage l DNA and of proteinase K digested-phenol extracted human mononuclear blood cell DNA do not show increase of electrophoretic mobility of them upon treatment with disulfide reducing agents, both gel electrophoretic and light scattering analyses of 3S and P point to the occurrence of particular nonhistone proteins that bind to particular sites of nuclear DNA in normal tissue cells by bonds resistant to 2.0 M NaCl and indicate that distant sites along the linear genomic sequence are physically linked by these proteins in a manner sensitive to disulfide reducing agents.

The light scattering and electrophoretic determinations and the fact that the disulfide reducing agents sensitive proteins and their effects are detected in normal cells lysed at ~ 2 °C in the presence of sulfhydryl blocking agents that block sulfhydryl oxidation and sulfhydryl-disulfide exchange reactions indicate that a subset of the nonhistone proteins that are undissociable from nuclear DNA by 2.0 M NaCl are undissociable also by 1 % SDS and have intermolecular and/or intramolecular disulfide bonds that are necessary for continued binding at particular sites of nuclear DNA in the presence of 1 % SDS.

Intermolecular S-S bonding of particular nonhistone proteins that are not dissociated from intact nuclear DNA by 2.0 M NaCl have in this respect been found by comparative SDS- PAGE analyses of samples under S-S reducing and nonreducing conditions. Among these nonhistone proteins, a protein with apparent molecular mass of ~ 220 kD of the monomeric form by SDS-PAGE has been determined to have dimers and multimers that are too large to enter into gels under nonreducing conditions and can be converted to monomeric form by treatment with 2-mercaptoethanol or dithiothreitol. Since redox state of this protein appeared to be targeted during neoplastic transformation in direction opposite to that found in normal tissue cells of aging organism and the protein has shown marked decrease of expression in neoplastic cells (Ta§ S, 1984), investigations were carried out to further characterize the protein and the particular DNA sites to which they are bound. Towards that, methods were developed that enable detection of structure of large DNA-protein complexes as they occur in cells.

Restriction endonucleases added into incubation medium of demembranized nuclei in tests like those described with DNAse I above make cuts to the DNA in chromatin in situ depending on whether or not a nucleotide sequence recognized by a restriction endonuclease is accessible to that enzyme. By performing restriction mapping of genomic DNA by this method in parallel with the conventional restriction mapping that uses proteinase K digested, phenol extracted nuclear DNA of a sample of the same cells, one can produce an accessibility map of genomic DNA in cells of interest and can determine how the conformation of chromatin changes in particular regions of genome during cellular differentiation, aging, neoplastic transformation and other processes. Enablement of these analyses by the availability of clones of numerous human genes throughout the human genome when they were initiated (circa 1986) has now been furthered by the availability of nucleotide sequence of essentially entire human genome. Likewise, methods to generate specific antibodies to a protein of interest detected by gel electrophoresis have been known. Generation of antibodies can start with excision of a gel region having the protein of interest. Such antibodies are useful for detection of the protein on blot membranes (e.g.“western blots”) and in tissues as well as in the cloning of genome sequences that encode for the protein to determine its amino acid sequence and structure. Identification of a particular DNA binding protein and antibodies to it facilitate also characterization of the sites (sequences of DNA) to which the protein binds, e.g. by capture and sequencing of the DNA fragments bound by the protein and by electrophoretic mobility assays that detect the bound and free forms of molecules. In the case of the aforementioned ~ 220 kD relatively abundant nuclear protein undissociable from DNA by 2.0 M NaCl, the protein has shown the unusual feature of being undissociable from DNA also by > 1% SDS. Because heating the sucrose density gradient purified nuclear DNA- tightly bound nonhistone protein complexes at 100 °C in gel loading buffers with SDS for SDS-PAGE causes denaturation of the double stranded DNA to single strands that undergo strand breaks during denaturation and pipetting, comparative analyses were done where the complexes in gel loading buffer with SDS were heated at 65 °C (maintains the DNA as double stranded; Ta§ S, 1990) and subjected to SDS-PAGE side by side with those heated at 100 °C along with samples predigested extensively with DNAse 1 prior to likewise SDS-PAGE. These analyses further confirmed that the protein showing ~ 220 kD apparent molecular mass of its monomeric form by SDS-PAGE is a DNA bound protein and has disulfide bonded dimeric and multimeric forms in normal cells.

Neoplastic cells typically do not show limitation of clonal lifespan in vitro and in vivo in serial transplantation assays in histocompatible laboratory animals unlike their normal counterparts. Since the number of divisions of normal cells prior to end of lifespan correlates with the species-specific maximum lifespan potential of donor animals and since the normal cells ceasing division display features that occur also in normal tissue cells with increasing age of animals, neoplastic cells are in general considered to escape from cellular aging. The unlimited lifespan potential of neoplastic cells fails on the other hand to represent a true escape from aging. In particular, the lowering of maintenance of genome integrity observed in the neoplastic cells of various primary tumors of human and experimental animals relative to that in their normal cells stands in sharp contrast to the elaborate measures to maintain genome integrity in germ line cells and during meiosis. Maintenance of integrity of genome involves measures against occurrence of damage to the genetic material from internal sources, such as particular byproducts of the oxidative energy metabolism and transposable sequences in genome, and from external sources such as ultraviolet and ionizing radiation, and involves repair of the damage that occurred. Structure of chromatin is a determinant of the repairability of damage to the genetic material and I have pointed previously that the heterochromatinization accompanying cellular differentiation during embryonal development and afterwards poses conflicts concerning repair of damage to the genetic material and that unrepaired damage is increased in cells of older animals in association with particular changes of chromatin structure. In view of the pointed out findings, I have carried out comparative analyses of normal and neoplastic cells by the methods described above.

Neoplastic lymphoid cells of human were compared with normal mononuclear blood cells of human with focus on the age associated changes of structure of chromatin.

Demembranized nuclei of these cells freshly prepared and incubated with DNAse I under identical conditions have shown significantly smaller average sizes of the 3S and P particles of the neoplastic cells than of normal cells in tests as described above (Table III). Significantly greater accessibility of the DNA in chromatin to DNAse I and to endogenous endonucleases was consistently found with the demembranized nuclei of neoplastic than normal cells of both young and older human subjects. Thus neoplastic cells did not appear to be closer to the normal cells of subjects of younger ages by this particular measure of chromatin structure. The neoplastic cells had even greater frequency of endonucleolytic cuts to the DNA in chromatin than of normal cells of older subjects.

A different situation was found on the other hand in comparisons of the same neoplastic cells with the same normal cells of young and older subjects by measurements of effects of disulfide reducing agents on structure of chromatin. Analyses of the DNA- protein complexes of 3S and P fractions with and without reduction of the disulfide bonds existing in the nuclei of the cells have shown significantly decreased average size of the 3S and P particles of normal cells upon reduction of the disulfide bonds while the 3S and P of neoplastic cells did not show a detectable effect under the same experimental conditions (Table III). By this measure the neoplastic cells were younger than normal cells in view of the findings that disulfide reducing agents have shown relatively greater effect on the nuclear lamina-matrix-DNA complexes of normal cells of the men and women in the > 50 years age group than in the 18-29 years interval (Table II). These results in determinations of effects of disulfide reducing agents on 3S and P particles in which DNA molecules are mostly bare except for the tightly DNA bound nonhistone proteins resistant to dissociation by 2.0 M NaCl accord with those described in Ta§ S et al, 1980b; Ta§ S, 1984 and Ta§ S et al, 1985b in comparisons of normal cells with their neoplastic counterparts by different methods and point to a relatively reduced redox state of the tightly DNA bound nonhistone proteins in neoplastic cells than in normal cells. They accord also with the markedly decreased existence of particular of these proteins in association with DNA of neoplastic cells than normal cells revealed by SDS-PAGE analyses of intact nuclear DNA-tightly bound nonhistone protein complexes purified by ultracentrifugation through neutral sucrose gradients (Ta§ S et al, 1980b; Ta§ S, 1984).

The above described significant modifications of structure of chromatin in normal tissue cells during aging and the targeting of them in neoplastic cells for modification in opposite direction are consistent with critical roles of them both in aging and in the escape of neoplastic cells from limitation of proliferative lifespan. The determinations for the first time of both the accessibility of DNA in chromatin and the effects of disulfide reducing agents on conformation of chromatin in both normal and neoplastic cells have in addition shown that neoplastic cells do not correct all age associated modifications of chromatin for being able to avoid the limited proliferative lifespan of normal cells. Redox state, expression and posttranslational modifications of particular DNA bound nonhistone proteins appear to be targeted in neoplastic cells in their escape from limitation of proliferative lifespan.

The described increases in the accessibility of DNA in chromatin and in the abundance of particular disulfide bonded proteins in nuclear lamina-matrix-DNA complexes in normal tissue cells with increase of age of organism are apparently related to responses to damaging of genetic material. Causation of accessibility of damaged DNA to repair enzymes and to cooperating proteins is a precondition of repair and involves multiple enzymatic modifications of chromatin proteins, e.g. poly(ADP-ribose) chain attachments that contribute to displacements of proteins from DNA and to chromatin unfolding. In order to maintain integrity of genome, not only the damaged nucleotides and strand breaks must be repaired without change of nucleotide sequence but the structure of chromatin in the region of damage and in the flanking sequences must also be reinstituted to the structure that existed prior to the damage. Provision of access to DNA by repair enzymes and by partner proteins can cause on the other hand accesses of other modifying proteins as well, e.g. of modifiers of methylation of DNA. In addition, causation of unfolding of heterochromatin carries the risk of activation of transposable elements when it occurs in regions having such elements. Genetic and epigenetic changes due to failures and errors of repair of DNA damage are particularly detrimental when they occur in stem cells and are found to increase with increase of age of organism despite the greater ability of normal stem cells than their differentiating progeny in prevention or decrease of damage to genetic material and in repair of such damage. Contrary to the normal stem cells that exist at particular locations in tissues (niches) where they are supported by niche cells, the neoplastic cells that develop during tumorigenesis and tumor progression have decrease of genetic stability and are selected to survive even in tissue environments other than where they have formed and typically show infinite lifespan potential. The findings described here show that these characteristics of neoplastic cells are associated with significant decrease of particular disulfide bonded nonhistone proteins that bind to particular sites of nuclear DNA and fold the nuclear DNA into loops which can also be tethered to elements of nuclear lamina-matrix. The described features and compromise in the maintenance of genome integrity in neoplastic cells lead to selection of the cells that allocate less and less resources for genetic stability and for differentiation so long as the mutations and epigenetic changes acquired are compatible with continued survival of them. Neoplastic cells that develop in this manner have selective advantages over normal tissue cells under the circumstances they form and generate tumors in tissues but are in general more vulnerable than normal cells when exposed to externally applied genotoxic agents in doses used in conventional genotoxic chemotherapy-radiotherapy of cancer. Genotoxic treatments of cancer patients are however ill-advised for multiple reasons, including the fact that genetic instability creates great genetic and phenotypic diversity in tumor cell populations to increase probability of occurrence of a clone of cancer cells resistant to an applied genotoxic agent to lead to relapse of tumor even after killing of the vast majority of cancer cells to cause non-detection of tumor in a patient by the imaging modalities used in clinical practice. The selective advantages that are conferred on neoplastic cells by their compromise of maintenance of genome integrity and means of avoiding differentiation can on the other hand be turned to their disadvantage by a nongenotoxic drug treatment that causes differentiation of them followed by their apoptosis to provide disappearance of tumors without relapse (Ta§ S, WO 2018/048367 Al).

The findings described here show that age of human subjects can be determined by analyses of cells obtained from them. Since cellular aging is at the roots of declining of multiple physiological functions in older individuals and is instrumental in occurrences of various age-associated diseases in human and since individuals show significant differences in such decline and occurrences, determining an indicator of biological age has utility in determining the rate of aging of individuals, in assessments of risks of diseases of aging and in preventive measures. The particular changes of structure of chromatin that are described here to occur with increase of age in human and to be causal in organismal aging can be determined quantitatively in cells taken from a person to obtain a measure of biological age and rate of aging of him or her. They can be determined by methods suitable for clinical laboratory practice as exemplified here. Comparisons of the scoring of individuals in tests by such methods relative to their calendar age provide an objective measure of health status and rate of aging of them. Reference values of a test for people in a given interval of calendar age, e.g. 50-55, 56-61 years and so on, or in broader or narrower intervals, can be determined as known in clinical laboratory practice. Plasma lipoproteins, for example, have descriptions of age dependent reference values. Specimens of over 120 subjects randomly sampled from a population are in general recommended to be analyzed to determine mean, median, 2.5th and 97.5th percentile values for reference intervals. The light scattering measurements described here have shown that such reference values for particular age groups can be readily determined and automation of the assays can provide data quickly from even larger populations for reference in comparisons of the measurement results of a given person. Identifications of critical determinants and molecular mechanisms of aging of human provide also guidance for interventions effective in prevention or delaying of diseases of aging and in treatments of them. The findings described here indicate that lowering or preventing damage to genetic material by internal and external sources and the repair of damage with accuracy without causation of the particular modifications of chromatin that are described here are effective measures against the age associated declining of physiological functions and against occurrences of diseases of aging. They show in the examples of neoplastic cells that occur commonly with increase of age that current practices that cause increase of damage to the genetic material are flawed for the particular reasons pointed and guide effective intervention with neoplastic diseases before and after occurrence.

Internal conflicts built in the human organism are instrumental in aging of human. Whereas human is one of the longest living species and the most intelligent of all species, it is clear that natural selection has its limits for improvements which are necessary if the predictable burden of aging and diseases of aging on individuals and human societies, worsening worldwide, is to be dealt with. The built in nature of the problem, the limits of the world for expansion of human population and the failures of simplistic approaches that include the symptomatic treatments of diseases of aging all point to a complex problem. Accurately identifying critical components that are at the origins of a complex problem serves on the other hand as basis for effective solutions and in this respect the findings described here do not indicate a problem that is impossible to solve. Further, although the human brain, the source of human intelligence, is also affected by aging, the mechanisms of cellular aging described here are operative in the cells of central nervous system as well and understanding of functioning of human brain has now advanced to effectively modulating various functions.

Statement: The previously undescribed experimental data that I describe here have been obtained in part in Kuwait where I was a faculty member at Kuwait University Faculty of Medicine during 1985-1990. War in Kuwait in 1990-1991 and the looting of the premises where 1 kept my laboratory data have caused losses of most except for those saved by one of my students and given to me after the war. I have been able to supplement the earlier analyses afterwards and they collectively form the basis of the findings described here. My expectations about laboratory facilities that did not materialize for the particular tests that I intended in relation to the findings described here contributed to the delay of description of the findings. Citations of publications here are restricted to those of my previous work that 1 consider most relevant to the findings described and are in general omitted where I consider that general knowledge of the scientists in the field of descriptions obviates citation. TABLES

Table L Measurements Of Light Scattering Of The Chromatin Fragments Generated By Endonucleolytic Cuts To DNA In Chromatin In Situ In Nucleus Of Mononuclear Blood Cells By Endogenous Endonucleases and By Limited Concentrations Of DNAse I Can Be Used To Detect Effects Of Aging In Human

Older/Young Age Group; Ratios Of Light Scatering Of Fragments Of

2S By Endogenous 3 S By Endogenous 2S By 3S By

Endonucleases Endonucleases DNAse I _ DNAse 1

0.63 ± 0.13 0.77 ± 0.15 0.68 ± 0.07 0.81 ± 0.06

Ratios of light scattering of the 2S and 3S fragments prepared from normal mononuclear blood cells of men and women in the > 50 years age group to those of the men and women in the 18-29 years range are shown for each fraction as the mean ± S.E.M. in > 9 separate experimental determinations wherein cells were obtained from 3-8 men and women of each age group in each experimental determination. Demembranized nuclei of cells were pooled for each age group in each experimental determination without discrimination of gender and concentrations of nuclei were equalized for the two age groups. Ratios for the fractions of nuclei incubated with DNAse I are for the nuclei incubated with 40-50 U/ml DNAse I for 5-30 minutes at ~ 23 or 37 °C in different determinations where the nuclei from young and older subjects were examined in parallel under identical conditions.

Ratios for the fractions of nuclei incubated without exogenously added endonuclease are for the nuclei incubated for 5-30 minutes at ~ 2 or ~ 23 or 37 °C. Ratios were smaller than 1.00 with the nuclei incubated under each indicated condition. The data are for the nuclei in native state, i.e. nuclei not treated with a disulfide reducing agent. All of the ratios are statistically significantly smaller than unity (1.00).

Table II. Measurements Of Light Scattering Of Nuclear Lamina-Matrix -DNA Complexes With and Without Reduction Of Disulfide Bonds Show Effects Of Aging In Human

Ratios Of Light Scattering Of The Complexes

Older/Y oung, Older A⁷ oung, S-S Reduced/Intact, S-S Reduced/Intact,

S-S Intact _ S-S Reduced _ Young _ Older _

0.88 ± 0.05 0.82 ± 0.08 0.90 ± 0.09 0.76 ± 0.04

Light scattering of complexes of normal mononuclear blood cells of the men and women in the > 50 years age group and those in the 18-29 years age range are shown as ratios of older/young age groups and ratios of light scattering of the complexes in which S-S bonds are reduced/nonreduced. The data are for complexes obtained by incubation of demembranized nuclei at ~ 2 °C or ~ 23 °C for ~ 5 minutes without exogenously added endonuclease (mean ± S.E.M.; pooled data of incubations at ~ 2 °C and ~ 23 °C).

p< 0.02, p<0.007, p<0.00003 in comparison to unity of ratio.

Table III. Measurements Of Light Scattering Of 3S Fragments and Of Nuclear Lamina- Matrix-DNA Complexes With and Without Reduction Of Disulfide Bonds Show That Conformation Of Chromatin Is Targeted By Neoplastic Transformation

Ratios Of Light Scattering Of The 3S Fragments and Of Complexes _

3S, Normal, 3S, Neoplastic, P, Normal, P, Neoplastic, 3S, P,

Reduced/Not Reduced/Not Reduced/Not Reduced/Not Neopl./Norm. Neopl./Norm.

0.77 ± 0.03^* 1.00 ± 0.08 0.93 ± 0.05^** 1.01 ± 0.13 0.83 ± 0.06^*** 0.82 ± 0.04^***

Light scattering of 3S fragments and of nuclear lamina-matrix-DNA complexes (P) of normal mononuclear blood cells of healthy human subjects and of neoplastic B lymphoid lineage cells (ARH-77) are compared between the 3S fragments and P in which S-S bonds were reduced or not (Reduced/Not ratios) and between 3S and P of neoplastic vs normal cells (Neopl./Norm. ratios). Demembranized nuclei of cells of subjects in 18-69 years age range were pooled in fractionations of nuclei in parallel with those of ARH-77 (mean ± S.E.M. of combined data of incubations of nuclei with 0-50 U/ml DNAse I for 5-30 minutes; > 3 separate determinations each). ^*p< 0.005, ^**p< 0.04, ^***p< 0.001 in comparison to unity of ratio.

Claims

1. A method of determining biological age and rate of aging of a human subject, comprising taking mononuclear blood cells or other cells from the subject and measuring accessibility of DNA in chromatin in nuclei of the cells and/or measuring degree of decondensation of chromatin upon treatment with disulfide reducing agents, and comparing the measurement results relative to reference values determined for persons who are in the same calendar age or in the same interval of calendar age as the subject.

2. A method according to claim 1 , wherein accessibility of DNA in chromatin is measured by measuring causation of endonucleolytic cuts to DNA by deoxyribonuclease 1 or by another endonuclease that has molecular mass of about 30 kilodalton or more and said decondensation is measured by measuring the degree of decrease of light scattering of the DNA-protein complexes that are obtained from the nuclei by endonucleolytic cuts to DNA and by dissociation of the DNA associated proteins from DNA by ionic strength of about 0.6 M or greater concentration of NaCl or KC1 or another salt.

3. A commercial package or diagnostic kit for use in determining biological age and rate of aging of a human subject, wherein the package or kit has one or more of the material used in a method as in claim 1 or 2 and has instructions of use as described in claim 1 or 2 and/or has regulatory approval for use as described in claim 1 or 2.