Effect of salt solutions on radiosensitivity of mammalian cells. III. Treatment with hypertonic solutions.

V79 Chinese hamster cells were treated with hypertonic solutions of NaCl or KCl and irradiated rat various times before, during, or after exposure to the solution. In solutions of molarities between 0-2 and 0-5 M, the cellular radiosensitivity increases with the molarity of the bathing solution. At these molarities, the hypertonic solution need not be present during irradiation to sensitize cells. Furthermore, radiosensitivity of cells could be increased by exposing cells for longer times to the hypertonic solution before irradiation. At higher salt concentrations (at 1-5 to 1-8 M), significant radioprotection is observed. Survival curve data showed that this protection was characterized by an increase in DO and a decrease in n, while the survival curves of cells sensitized with 0-465 M NaCl or with lower concentrations exhibited mainly changes in DO. The 1-55 M NaCl solution must be present during radiation to give a protective effect. Prolonged exposure to the salt before irradiation reduced the amount of radioprotection afforded by the salt. The results are discussed in terms of the effects of ions on histones, cellular water structure and the cell-aging cycle.     

The effects of various salt and sucrose solutions on the U.V.L. sensitivity of CHO cells.

The response of cultured CHO cells to U.V.L. irradiation during treatment with anisotonic solutions shows that treatment with hypotonic sucrose, NaCl or KCl solutions causes an increase in the cellular U.V.L. sensitivity, while exposure to hypertonic solutions causes a large decrease in U.V.L. sensitivity. Cells exposed to 1.8 M sucrose, NaCl or KCl solutions and given a U.V.L. dose of 252 erg/mm2 towards the end of the 20 min solution exposure time have survival levels which are respectively 228,26, and 23 times higher than the controls, i.e. cells irradiated in phosphate buffered saline. Cell volume data obtained using a Coulter counter, and nuclear area data of attached cells obtained using an optical microscope with a micrometer reticle, show that cell and nuclear size are related to U.V.L. sensitivity. That is, as cells shrink and the nuclear area decreases, the cells become more U.V.L.-resistant. During hypotonic treatment with 0.1 M NaCl, the cell volume, nuclear area and U.V.L. sensitivity increased in the first 2 to 4 min of exposure time, but at longer exposure times (greater than 3 to 4 min), cell volume, nuclear area and cellular U.V.L. sensitivity decreased. For 0.1 M KCl treatment the cells initially displayed a rapid increase in volume, nuclear area and U.V.L. sensitivity, but at the longer exposure times no decrease in cell and nuclear size were observed, and a slight increase in U.V.L. sensitivity occurred. Changes in U.V.L. sensitivity were related to changes in nuclear size and cell volume; however, calculations showed that during hypertonic treatment there is an ionic effect as well as an osmotic effect. That is, the cellular U.V.L. survival in equal hypertonic concentrations of NaCl or KCl was lower than in the same concentration of sucrose.     

Experimental visualization of chromoneme as one of the higher levels of chromatin compactization in the mitotic chromosome

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1-0.2 microm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoeneme structure of the mitotic chromosomes in the presence of Ca2+. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl2. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosome swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca2+ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl2, were treated with a photosensbilizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and their general pattern of packaging in in the chromatic was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution, which normally leads to chromoneme destruction. An even more rigid treatment of the stabilized chromosomes with a 2 M NaCl solution, which normally fully decondenses the chromosomes, made it possible to detect a 3D reticular skeleton devoid of any axial structures.     

Recovery of the viability of Chinese hamster V79-4 cells after combined exposure to hyperthermia and radiation

The survival of cells overheated (42 degrees C) before gamma irradiation is increased by holding them in the growth medium at 37 degrees C before treatment with hypertonic NaCl solution. The substantial synergistic effect of hyperthermia and radiation takes place when the cells are treated with a 1.5 M NaCl solution immediately after the combined action of these inactivating factors. The synergistic effect is decreased by holding the cells in the nutrient medium at 37 degrees C for 4 hours before hypertonic treatment.     

Fixation of potentially lethal radiation damage by post-irradiation exposure of Chinese hamster cells to 0.5 M or 1.5 M NaCl solutions.

The effect of 0.05 M and 1.5 M NaCl treatments on CHO cells during and after irradiation has been examined. Treatment with either hypotonic or hypertonic salt solutions during and after irradiation resulted in the fixation of radiation damage which would otherwise not be expressed. The half time for fixation was 4 to 5 min, and the increased expression of the potentially lethal damage by anisotonic solutions was mainly characterized by large decreases in the shoulder of the survival curve, as well as by decreases in DO. Fixation of radiation damage at 37 degrees C occurred to a much greater extent for the hypertonic treatment than for the hypotonic treatment and was greater at 37 degrees C than at 20 degrees C. Although both the hypotonic and hypertonic treatments during and after irradiation reduced or eliminated the repair of sublethal and potentially lethal damage, treatment during irradiation only, radiosensitized the cells when the treatment was hypotonic, and radioprotected the cells when the treatment was hypertonic. These observations are discussed in relation to salt treatments and different temperatures altering competition between repair and fixation of potentially lethal lesions, the number of which depends on the particular salt treatment at the time of irradiation.     

Experimental Visualization of Chromoneme as a Higher Level of Chromatin Compactization in the Mitotic Chromosome

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1–0.2 μm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoneme structure of the mitotic chromosomes in the presence of Ca²⁺. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl₂. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosomes swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca₂₊ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl₂, were treated with a photosensitizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and the general pattern of their packaging in the chromatid was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution, which normally leads to chromoneme destruction. An even more rigid treatment of the stabilized chromosomes with a 2 M NaCl solution, which normally fully decondenses the chromosomes, made it possible to detect a 3D reticular skeleton devoid of any axial structures. __________ Translated from Ontogenez, Vol. 36, No. 5, 2005, pp. 323–332. Original Russian Text Copyright ? 2005 by Burakov, Tvorogova, Chentsov.     

Deficiency in fast repair process of potentially lethal damage induced by X-irradiation in fibroblasts derived from LEC strain rats.

The time course for the repair of PLD in LEC and WKAH rat cells irradiated at 5 Gy was examined. In the case of WKAH rat cells, the surviving fraction increased with increasing incubation times after X-irradiation. When hypertonic treatment was performed at each incubation time with 0.5 M NaCl for 20 min, increase in the surviving fractions was not shown. In contrast, no significant recovery of the surviving fraction in LEC rat cells was observed after incubation of irradiated cells with or without 0.5 M NaCl for 20 min. On dose-survival curves, hypertonic treatment with 0.5 M NaCl enhanced radiosensitivity of WKAH rat cells, but not LEC rat cells. Although the surviving fraction of the cells from backcross mice with normal radiosensitivity reduced by treatment with 0.5 M NaCl, the survival fraction was not affected in the cells from backcross mice with higher radiosensitivity by treatment with 0.5 M NaCl. When the cells were X-irradiated and incubated with or without 0.225 M NaCl, the radiosensitivities of LEC and WKAH rat cells treated with 0.225 M NaCl for 4 h were approximately two-fold higher than those of untreated cells. Treatment with caffeine also reduced the surviving fractions of both X-irradiated LEC and WKAH rat cells, compared with those of untreated cells. These results indicated that the slow repair of PLD occurred in LEC rat cells but not the fast repair of PLD.     

The integrity of the histone-DNA complex in chromatin fibres is not necessary for the maintenance of the shape of mitotic chromosomes

In this study we addressed the question of whether scaffold structures produced from purified mitotic chromosomes are an artefact of dehistonization, and whether the integrity of the chromatin fibres is necessary for the maintenance of the well-known shape of mitotic chromosomes. Purified mitotic chromosomes from Friend erythroleukemia cells were treated either with increasing NaCl concentrations up to 500 mM, or with 6 M urea in the presence or absence of 10 mM 2-mercaptoethanol. The main criterion for the intactness of the overall chromosome shape as seen by electron microscopy was the characteristic X-or U-like appearance with clearly discernable chromatid axes. Histone H1 is known to be essential for the integrity of chromatin fibres. Its removal in sucrose gradients containing 500 mM NaCl did not lead to loss of the overall chromosome shape. However, treatment of chromosomes in sucrose gradients containing 10 mM 2-mercaptoethanol and 6 M urea led to loss of the structure probably due to dissociation (or denaturation) of shape-determining (scaffolding) components. Under these conditions most of the histones remained bound to the chromosomes, and the fibres in this chromatin material, after removal of excess urea and 2-mercaptoethanol, still showed condensation of the nucleosome filaments into the characteristic fibre structures upon increasing ionic strength. Our observations are compatible with the model that specific non-histone components, independently of histone-DNA interactions, organize or stabilize the structure of metaphase chromosomes.     

Hypertonic treatment inhibits radiation-induced nuclear translocation of the Ku proteins G22p1 (Ku70) and Xrcc5 (Ku80) in rat fibroblasts

The effects of X irradiation and hypertonic treatment with 0.5 M NaCl on the subcellular localization of the Ku proteins G22p1 (also known as Ku70) and Xrcc5 (also known as Ku80) in rat fibroblasts with normal radiosensitivity were examined using confocal laser microscopy and immunoblotting. Although these proteins were observed mainly in the nuclei of human fibroblasts, approximately 80% of the intensities of immunofluorescence from both G22p1 and Xrcc5 was observed in the cytoplasm of rat fibroblasts. When the rat cells were X-irradiated with 4 Gy, the intensities of the fluorescence derived from G22p1 and Xrcc5 in the nuclei increased from 20% to 50% of the total cellular fluorescence intensity at 20 min postirradiation. No significant differences were observed between the total intensities of the cellular fluorescence from the proteins in unirradiated and irradiated rat fibroblasts. The results showed that the proteins were translocated from the cytoplasm to the nucleus in the rat cells after X irradiation. The nuclear translocation of the proteins from the cytoplasm was inhibited by hypertonic treatment of the cells with 0.5 M NaCl for 20 min, which inhibits the fast repair process of potentially lethal damage (PLD). When the rat cells were treated with 0.5 M NaCl immediately after X irradiation, the repair of DNA DSBs was inhibited. The surviving fraction was approximately 60% of that of irradiated cells that were not treated with 0.5 M NaCl. The surviving fraction increased with incubation time in the growth medium before treatment with NaCl. The proportions of the intensities of fluorescence from G22p1 in the nuclei of X-irradiated cells also increased from 20% to 50% with increasing interval between X irradiation and treatment with NaCl. These results suggest that nuclear translocation of G22p1 and Xrcc5 is important for the fast repair process of PLD in rat cells.     

Lamins A and C bind and assemble at the surface of mitotic chromosomes

To study a possible interaction of nuclear lamins with chromatin, we examined assembly of lamins A and C at mitotic chromosome surfaces in vitro. When a postmicrosomal supernatant of metaphase CHO cells containing disassembled lamins A and C is incubated with chromosomes isolated from mitotic Chinese hamster ovary cells, lamins A and C undergo dephosphorylation and uniformly coat the chromosome surfaces. Furthermore, when purified rat liver lamins A and C are dialyzed with mitotic chromosomes into a buffer of physiological ionic strength and pH, lamins A and C coat chromosomes in a similar fashion. In both cases a lamin-containing supramolecular structure is formed that remains intact when the chromatin is removed by digestion with micrococcal nuclease and extraction with 0.5 M KCl. Lamins associate with chromosomes at concentrations approximately eightfold lower than the critical concentration at which they self-assemble into insoluble structures in the absence of chromosomes, indicating that chromosome surfaces contain binding sites that promote lamin assembly. These binding sites are destroyed by brief treatment of chromosomes with trypsin or micrococcal nuclease. Together, these data suggest the existence of a specific lamin-chromatin interaction in cells that may be important for nuclear envelope reassembly and interphase chromosome structure.     

DNA-binding proteins from Novikoff hepatoma cells.

In 0.05 M NaCl, 6-8% of the total soluble proteins from Novikoff hepatoma cells bind rapidly and reversibly to columns containing either heterologous or homologous DNA adsorbed to cellulose. These proteins can be eluted by buffer containing 2.0 M NaCl. 0.5-1% of the total protein exhibits a 7-17-fold preference for rat DNA over Escherichia coli DNA. 1-1.5% of the proteins bind DNA so strongly that elution cannot be effected by 4.0 M NaCl but can be accomplished by deoxyribonuclease I treatment of the columns. DNA-binding proteins eluted by 2.0 M NaCl were labeled with 125I or 131I and characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. These experiments indicate that DNA-binding proteins represent a discrete subset of the total soluble protein. Many similarities were noted between the major components of the homologous and heterologous DNA-binding fractions.     

Modification of cell survival and transformation by exposure to anisotonic solutions during irradiation

C3H 10T 1/2 cells were exposed to a wide range of anisotonic NaCl solutions and were irradiated during the last few minutes of the salt treatment. Radiosensitization in terms of cell killing and transformation was observed for hypotonic NaCl treatment. Hypertonic treatment with 0.5 mol/liter NaCl also caused radiosensitization for cell killing and transformation, while extreme hypertonic treatment (1.5 mol/liter) resulted in radioprotection for both end points. Radiosensitization for both end points declined as the hypotonic treatment (0.05 mol/liter) was prolonged. To a lesser extent, a decline in radioprotection by 1.5 mol/liter treatment occurred as exposure time increased.     

Early chromosome condensation without cell fusion. I. Preliminary results with allogeneic and xenogeneic cells.

Early chromatin condensation in interphase cells (G1) of human peripheral blood lymphocytes has been induced without virus or cell fusion by exposure to allogeneic or xenogeneic mitotic cells. The event, although similar in some ways to the phenomenon described as "premature chromosome condensation," "chromosome pulverization," and "prophasing," differs in that it does not require the presence of viruses and cell fusion before mitosis proceeds in the G1 cell. Early chromatin condensation in interphase cells induced by mitotic cells only, consists of chromatids in the early or late G1 phase of the cell cycle that are not pulverized or fragmented at mitosis. Some of the chromosomes are twice as long as the metaphase chromosomes and exhibit natural bands. Almost twice as many of these bands are produced as by trypsin treatment of metaphase chromosomes. The nuclear membrane is intact and nucleoli are present, to which some chromosomes are attached. The DNA content of the precocious chromosomes in G1 is half the amount of the metaphase complement.     

Common pathway of chromosome condensation in Mammalian cells

Chromatin folding in the interphase nucleus is not known. We compared the pattern of chromatin condensation in Indian muntjac, Chinese hamster ovary, murine pre B, and K562 human erythroleukemia cells during the cell cycle. Fluorescent microscopy showed that chromosome condensation follows a general pathway. Synchronized cells were reversibly permeabilized and used to isolate interphase chromatin structures. Based on their structures two major categories of intermediates were distinguished: (1) decondensed chromatin and (2) condensed chromosomal forms. (1) Chromatin forms were found between the G1 and mid-S phase involving veil-like, supercoiled, fibrous, ribboned structures; (2) condensing chromosomal forms appeared in the late-S, G2, and M phase, including strings, chromatin bodies, elongated pre-chromosomes, pre-condensed chromosomes, and metaphase chromosomes. Results demonstrate that interphase chromosomes are clustered in domains; condensing interphase chromosomes are linearly arranged. Our results raise questions related to telomer sequences and to the chemical nature of chromosome connectivity.     

A search for protein cores in chromosomes: Is the scaffold an artifact?

A protein chromosome scaffold structure has been proposed that acts as a structural framework for attachment of chromosomal DNA. There are several troubling aspects of this concept: (1) such structures have not been seen in many previous thin-section and whole-mount electron microscopy studies of metaphase chromosomes, while they are readily seen in leptotene and zygotene chromosomes; (2) such a structure poses problems for sister chromatid exchanges; and (3) the published photographs show a marked variation in the amount of scaffold in different whole-mount preparations. An alternative explanation is that the scaffold in whole-mount preparations represents incomplete dispersion of the high concentration of chromatin in the center of chromosomes, and when the histones are removed and the DNA dispersed, the remaining nonhistone proteins (NHPs) aggregate to form a chromosome-shaped structure. Two studies were done to determine if the scaffold is real or an artifact: (1) Chinese hamster mitotic cells and isolated chromosomes were examined using two protein stains -EDTA-regressive staining and phosphotungstic acid (PTA) stain. The EDTA-regressive stain showed ribonucleoprotein particles at the periphery of the chromosomes but nothing at the center of the chromosomes. The PTA stain showed the kinetochore plates but no central structures; and (2) isolated chromosomes were partially dispersed to decrease the high concentration of chromatin in the center of the chromosome, then treated with 4 M ammonium acetate or 2 M NaCl to dehistonize them and disperse the DNA. Under these circumstances, no chromosome scaffold was seen. We conclude that the scaffold structure is an artifact resulting from incomplete dispersion of central chromatin and aggregation of NHPs in dehistonized chromosomes.     

Recovery from thermal damage of mammalian cells fixed by treatment with a hypertonic sodium chloride solution

The survival of Chinese hamster cell V79-4 after hyperthermic treatment (42 degrees C, 40 minutes) in the exponential growth phase considerably increases with the duration of holding them in the growth medium at 37 degrees C before hypertonic salt treatment (1.5 M NaCl, 15 minutes). The experimental data are interpreted as a recovery of mammalian cells from thermal lesions, whose lethal action manifests itself at high salt concentrations.     

Differential repair of potentially lethal damage in exponentially growing and quiescent 9L cells

The alteration of potentially lethal damage repair by postirradiation treatment with hypertonic saline (0.5 M PBS) was investigated in exponentially growing and quiescent 9L cells in vitro. A single dose of X rays (8.5 Gy) immediately followed by a 30-min treatment with hypertonic PBS at 37 degrees C reduced the survival of exponentially growing 9L cells by a factor of 13-18 compared to survival of irradiated immediately and delayed-plated cells, while the survival of quiescent cells was reduced by only a factor of 5-8. Survival curves confirmed the relative resistance of the quiescent 9L cells versus exponentially growing 9L cells to X rays plus hypertonic treatment. Both the slope and the shoulder of the survival curve were reduced to a greater extent in exponentially growing cells than in the quiescent cells by hypertonic treatment. The response of quiescent cells cannot be explained by either the duration of hypertonic treatment or the redistribution of the cells into G1 phase. We show that quiescent 9L cells can recover from hypertonically induced potentially lethal damage when incubated under conditions which have been found to delay progression through the cell cycle, and postulate that an altered chromatin structure or an enhanced repair capacity of quiescent 9L cells may be responsible for their resistance.     

Recovery from potentially lethal damage and recruitment time of noncycling clonogenic cells in 9L confluent monolayers and spheroids

Cells that have been grown as multicell tumor spheroids exhibit radioresistance compared to the same cells grown in monolayers. Comparison of potentially lethal damage (PLD) repair and its kinetics was made between 9L cells grown as spheroids and confluent monolayers. Survival curves of cells plated immediately after irradiation showed the typical radioresistance associated with spheroid culture compared to plateau-phase monolayers. The dose-modification factor for spheroid cell survival is 1.44. Postirradiation incubations in normal phosphate-buffered saline (PBS), conditioned media, or 0.5 M NaCl in PBS reduced the differences in radiosensitivity between the two culture conditions. Postirradiation treatment in PBS or conditioned medium promoted repair of potentially lethal damage, and 0.5 M NaCl prevented the removal of PLD and allowed the fixation of damage resulting in lower survival. Survival of spheroid and monolayer cells after hypertonic NaCl treatment was identical. NaCl treatment reduced Do more than it did the shoulder (Dq) of the survival curve. PLD repair kinetics measured after postirradiation incubation in PBS followed by hypertonic NaCl treatment was the same for spheroids and for plateau-phase monolayers. The kinetics of PLD repair indicates a biphasic phenomenon. There is an initial fast component with a repair half-time of 7.9 min and a slow component with a repair half-time of 56.6 min. Most of the damage (59%) is repaired slowly. Since the repair capacity and kinetics are the same for spheroids and monolayers, the radioresistance of spheroids cannot be explained on this basis. Evidence indicates that the time to return from a Go (noncycling G1 cells) state to a proliferative state (recruitment) for cells from confluent monolayers and from spheroids after dissociation by protease treatment may be the most important determinant of the degree of PLD repair that occurs. Growth curves and flow cytometry cell cycle analysis indicate that spheroid cells have a lag period for reentry into a proliferative state. Since plating efficiency remains high and unchanging during this period, one cannot account for the delay on the basis of the existence of a large fraction of Go cells which are not potentially clonogenic. The cell cycle progression begins in 6-8 h for monolayer cells and in 14-15 h for spheroids. It is hypothesized that the slower reentry of spheroid cells into a cycling phase allows more time for repair than for the rapidly proliferating monolayer cells.     

Mevalonate deprivation leads to aneuploidy in Chinese hamster ovary cells

After exposure to compactin, the competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 22% of CHO-K1 cells contained abnormally high numbers of chromosomes. In two populations of cells selected for compactin resistance 31 and 33% of the cells contain more than 22 chromosomes. Some cell lines isolated from these populations have the wild type chromosome number of 20-21, while others have a broad distribution of chromosome number, often with a mean around 36-40. Finally, Chinese hamster ovary cells that are mutant for 3-hydroxy-3-methylglutaryl-CoA reductase and therefore auxotrophic for mevalonate were starved for that compound. This treatment also increased the number of cells containing extra chromosomes. These results indicate that interruption of the cellular supply of mevalonate results in abnormal chromosome number.     

Nuclear matrix proteins are carried within peripheral material of mitotic chromosomes

Immunofluorescent analysis has shown that autoimmune sera M-222 and M-260 are bound to interphase nuclei and mitotic chromosomes of the pig embryo kidney cell culture. The fluorescent stain is diffuse in nuclei and forms a thin fluorescent area around each nucleolus, whereas the nucleolar cores are unstained. The periphery of each mitotic chromosome is stained distinctly. After removal of histones and DNA by the cell treatment with 2 M NaCl and DNase I, the Hoechst 33258 staining of nuclei and chromosomes disappears completely, whereas the pattern of staining with antibodies is not changed as compared with normal cells. Electron microscopy revealed in interphase nuclei after such treatment only lamina, residual nucleoli, and the intranuclear matrix network, and antibodies are bound just to these elements. Molecular mass of proteins bound to these antibodies was determined by immunoblotting. Serum M-260 contained antibodies to a single 65 kDa polypeptide, whereas antibodies to two polypeptides of 47 and 65 kDa were found in M-222. After chromatin removal and revealing nuclear protein matrix, M-222 binds only to 65 kDa polypeptides. Thus, peripheral chromosomal material is involved in transfer of the nuclear matrix polypeptide to daughter nuclei during mitosis.