ARTICLE |
Correspondence to: James M. Jamison, Dept. of Urology, Northeastern Ohio Universities College of Medicine, 4209 State Route 44, PO Box 95, Rootstown, Ohio 44272-0095. E-mail: jmj@neoucom.edu
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Summary |
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Human prostate cancer cells (DU145) implanted into nude mice are deficient in DNase activity. After administration of a vitamin C/vitamin K3 combination, both alkaline DNase (DNase I) and acid DNase (DNase II) activities were detected in cryosections with a histochemical lead nitrate technique. Alkaline DNase activity appeared 1 hr after vitamin administration, decreased slightly until 2 hr, and disappeared by 8 hr after treatment. Acid DNase activity appeared 2 hr after vitamin administration, reached its highest levels between 4 and 8 hr, and maintained its activity 24 hr after treatment. Methyl green staining indicated that DNase expression was accompanied by a decrease in DNA content of the tumor cells. Microscopic examination of 1-µm sections of the tumors indicated that DNase reactivation and the subsequent degradation of DNA induced multiple forms of tumor cell death, including apoptosis and necrosis. The primary form of vitamin-induced tumor cell death was autoschizis, which is characterized by membrane damage and the progressive loss of cytoplasm through a series of self-excisions. These self-excisions typically continue until the perikaryon consists of an apparently intact nucleus surrounded by a thin rim of cytoplasm that contains damaged organelles. (J Histochem Cytochem 49:109119, 2001)
Key Words: vitamin C, vitamin K3, DNase, prostate cancer, necrobiology, cancer cell death
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Introduction |
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Prostate carcinoma is one of the most prevalent malignant tumors of the male reproductive organs, with an estimated 179,300 cases and 37,000 deaths reported annually (
The results of previous studies demonstrate that the activity of alkaline DNase (DNase I; EC 3.1.21.1) and acid DNases (DNase II; EC 3.1.22.1) is inhibited in non-necrotic cells of malignant tumors in men and in experimental animals, as well as during the early stages of experimental carcinogenesis (
The tumor growth-inhibiting and -chemosensitizing effects of the vitamin C and K3 combination were confirmed by in vitro experiments with different lines of human tumor cells (
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Materials and Methods |
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Tumor Cells
The human androgen-independent prostate carcinoma DU145 cell line was obtained from the American Type Culture Collection (Rockville, MD) and was grown in McCoy's 5A medium (M5A; Gibco, Grand Island, NY). All media were supplemented with 10% fetal bovine serum (FBS; Gibco), 2.2 g/liter NaHCO3 (Gibco), and 50 µg/ml gentamicin sulfate (Fisher Scientific; Pittsburgh, PA). Cells were grown as adherent cells in a humidified atmosphere at 37C in 5% CO2. When the tumor cells were used for experiments, they were harvested with trypsin-ethylenediamine tetraacetic acid (EDTA) (JRH Biosciences; Lenexa, KS), washed with PBS, pH 7.4, and resuspended in M5A. Cells were counted with a hemocytometer using trypan blue (Gibco) exclusion to determine the number of viable cells.
Reagents
Vitamin C (VC) and menadione bisulfite (VK3) were purchased from Sigma Chemical (St Louis, MO) and were dissolved in PBS to create oral vitamin doses of VC = 15 g/liter and VK3 = 0.15 g/liter. The IP doses of the vitamins were VC = 1 g/kg body mass and VK3 = 10 mg/kg body mass as optimized and described by Taper and co-workers (1987). All vitamins were prepared in a darkened laminar flow hood to prevent photo-inactivation.
Animals
Male athymic nude mice (NCr-nu/nu; 4 weeks old) were purchased from Taconic Farms (Germantown, NY) and maintained in microinsulator cages (within the AALAC-accredited NEOUCOM Comparative Medicine Unit) in a pathogen-free isolation facility. After a 1-week isolation period, 1 x 106 DU145 cells in a 100-µl volume of M5A medium were injected SC into the median and dorsal scapular region. After 4 weeks, the mice were weighed and divided into six groups of four animals each. Five of the six groups were given 100 µl of the VC/VK3 combination by gavage and then immediately given an additional 100-µl dose of the vitamin combination by IP injection. Control mice received two 100-µl doses of PBS. The mice were sacrificed by carbon dioxide inhalation at 1, 2, 4, 8, or 24 hr after vitamin administration. The tumors were then surgically removed and trisected. One third of the tumor was flash-frozen in liquid nitrogen and stored at -80C. The second third of the tumor was fixed in Carnoy's fixative for 3 hr, processed, and embedded in paraffin. The final third of the tumor was fixed in buffered glutaraldehyde and processed in epoxy resin for future electron microscopic observations as described below.
All animal procedures were performed under protocols approved by the Northeastern Ohio Universities College of Medicine Animal Care and Use Committee.
Plastic Sections
One third of each tumor from vitamin-treated and sham-treated mice was fixed in buffered glutaraldehyde solution (3.2% in 0.1 M phosphate buffer, pH 7.32) and processed for ultrastructural observations in epoxy resin (Polysciences, PolyBed 812; Warrington, PA) (
Histochemical Detection of DNase Activity
The histochemical activity of alkaline and acid DNases was detected in cryosections of the frozen tumor specimens using a modified Gomori's lead nitrate method (
Histochemical Detection of Nucleic Acids
Nucleic acid staining was performed using the classic methyl greenpyronin technique of
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Results |
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Table 1 and Fig 1 summarize the results of the histochemical determination of alkaline and acid DNase activity in the tumors at different time intervals after single-dose administration of IP and oral vitamins C and K3 or PBS administration. As shown in Table 1, the sham-treated tumors exhibited little if any alkaline and acid DNase activity. However, after combined vitamin C and K3 treatment, alkaline DNase activity appeared sooner than the acid DNase activity. This positive alkaline DNase activity was significant, appeared as early as 1 hr after vitamin administration, and decreased slightly until 2 hr after vitamin treatment. Alkaline DNase activity gradually diminished until 8 hr after vitamin administration and then disappeared. Acid DNase activity appeared 2 hr after vitamin administration, reached its highest level 48 hr after treatment, and maintained its activity 24 hr after treatment.
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Fig 1 is a montage of representative light micrographs of tumor sections that were stained for alkaline or acid DNase activities. One hour after combined vitamin C and K3 injection, alkaline DNase demonstrated distinct activity in the cytoplasm. However, the majority of alkaline DNase activity was found in the nuclei of tumor cells (Fig 1, Fig 1h). As described in Table 1, alkaline DNase activity gradually decreased and was not evident after 4 hr. Very low to undetectable alkaline DNase activity was seen in the sham tumor cells (Fig 1C). Conversely, little acid DNase activity was observed until 4 hr after vitamin treatment. At this time, intense acid DNase activity was detected primarily in the nuclei of the tumor cells (Fig 1, Fig 4h). Acid DNase activity gradually decreased. Substantial activity was still evident 24 hr after vitamin treatment. Sham-treated tumor cells exhibited little acid DNase activity (Fig 1C).
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The consequence of the reactivation of alkaline and acid DNases could be seen in tumor cells 24 hr after vitamin C and K3 treatment. When the nucleic acids of sham-treated tumor cells were stained with methyl greenpyronin Y, intense methyl green staining was visible in the nucleus and intense pyronin Y staining was observed in the cytoplasm (Fig 2A). In vitamin-treated tumor cells (Fig 2B), the methyl green staining was greatly diminished, indicative of a decrease in DNA content. Densitometric analysis of the staining intensity of control and vitamin-treated tumor cells produced optical density values of 0.63 ± 0.11 and 0.37 ± 0.07, respectively. These values indicate a statistically significant (p<0.01) decrease in DNA staining. Pyronin Y staining diminished at a more rapid rate and to an even greater extent than the methyl green staining, indicative of decreased RNA content. This suggests that this combined vitamin treatment may also reduce transcription or induce the activation of RNases.
Fig 3 is a set of micrographs of hematoxylin- and eosin-stained sections of sham-treated and vitamin-treated tumors 4 hr after treatment. The cells in the sham-treated tumor (Fig 3A) exhibited pleomorphic cellular and nuclear morphology. The presence of mitotic figures indicated that cells in this tumor were still actively dividing. Sections of the control tumor exhibited an average of 5 ± 1 mitotic figures/500 cells. Conversely, the cells in the vitamin-treated section (Fig 3B) averaged 1.23 ± 0.84 mitotic figures/500 cells. Although the incidence of mitosis was small, the difference between control and vitamin-treated cells was statistically significant (p<0.01). The nuclei of some cells appeared pyknotic, whereas other cells contained nuclei with marginated chromatin and predominant condensed nucleoli. This section was also characterized by the presence of many vacuolated cells and the presence of necroses.
Fig 4 Fig 5 Fig 6 is a gallery of light micrographs of Toluidine blue-stained sections of vitamin-treated tumors 4 hr after treatment, depicting tumor cells in various stages of cell injury and cell death. These micrographs are characteristic of this tumor and reveal a plain, compact mass with few capillaries and little to no mitotic activity. The poor vascularization of these tumors (v = 250 mm3) is probably not a function of vitamin treatment because angiogenesis in tumors derived from DU145 cells is greatly diminished compared to that of other prostate cancer cell lines (
Fig 5 and Fig 6 display similar but more exacerbated cell changes that result in multiple types of cell death, including necrosis, apoptosis, and autoschizis. Whereas necrosis, apoptosis, and autoschizis were not observed in control tumor cells, an average of 9.0 ± 2.1 autoschizic, 3.0 ± 1.1 necrotic, and 1.97 ± 0.45 apoptotic cells were observed per 500 cells after vitamin treatment. These results are consistent with the results obtained with annexin Vpropidium iodide-labeled cells (
The process of autoschizis is more evident in Fig 6, in which the vacuolizations are marked by stars and the cytoplasmic excisions are labeled with arrowheads, with intercellular vacuolizations and exaggerated intercellular spaces (i). These autoschizic tumor cells were found lining the necrotic foci of the tumor, which often form around capillaries such as the one in the center of Fig 6. These cells are characterized by exaggerated membrane damage and by a process of progressive loss of cytoplasm in which the perikaryal compartment separates from the main cytoplasmic cell body in a series of self-excisions. These self-excisions (dark arrowheads with short shafts) typically continue until the perikaryon consists of an apparently intact nucleus and a thin rim of cytoplasm containing damaged organelles. The excised cytoplasmic blebs are primarily filled with dense glycogen patches but contain no organelles or nuclear fragments. These cells are characterized by exaggerated membrane damage and by progressive loss of cytoplasm in which the perikaryal compartment separates from the main cytoplasmic cell body in a series of self-excisions. During autoschizis, nucleoplasm becomes more chromatic and nuclear size decreases. Therefore, the size of the resultant autoschizic cell or body is much smaller than the tumor cell from which it originated.
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Discussion |
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Because endonuclease activation is one of the earliest changes denoting irreversible commitment to cell death, it is generally believed to be involved in the triggering of cell death rather than to be the result of it. Several candidate deoxyribonuclease molecules have been identified in various cell lines and tissues, including the caspase-activated DNA fragmentation factor caspase-3-activated DNase (DFF40/CAD) nuclease, DNase I, and DNase II. DFF endonuclease is primarily responsible for mediating DNA laddering during apoptosis after its activation by caspases (mainly caspase 3). DFF is composed of two subunits, a 40-kD caspase-3-activated nuclease and its 45-kD inhibitor. This protein complex resides in the nucleus and is activated by caspase-3 cleavage of the inhibitor and its subsequent dissociation from the endonuclease. The activated endonuclease exhibits a pH optimum of 7.5, requires Mg2+ (not Ca2+), and is inhibited by Zn2+ (
Two of these DNases (DNase I and DNase II) are of particular interest because of their broad tissue distribution and because they have been implicated as possible effectors of apoptosis (
In contrast to DNase I, DNase II (acid DNase) does not require divalent cations for its activity (
The activity of alkaline and acid DNases is inhibited in non-necrotic cells of malignant tumors in humans and experimental animals (
The compounds that reactivate alkaline and acid DNase activities demonstrate a distinct enzymatic specificity (
Although the relatively rapid reversiblity of the deficiency of alkaline DNase argues against a real decrease in enzyme content and the subsequent de novo synthesis of the DNase, the later time course of acid DNase activity could involve activation of both pre-existing and de novo acid DNase synthesis (
Protein nuclease inhibitors that bind to DNase molecules in a 1:1 ratio have been found in normal tissues (
The results of the current study demonstrate that sham-treated tumor cells are essentially devoid of DNase activity. After administration of a vitamin C/Vitamin K3 combination, both alkaline DNase (DNase I) and acid DNase (DNase II) activity was detected in cryosections of the frozen tumor specimens by a modified Gomori's lead nitrate method. Alkaline DNase activity appears as early as 1 hr after vitamin administration, decreases until 2 hr, and disappears by 8 hr after vitamin administration. Acid DNase activity appears 2 hr after vitamin administration, reaches its highest level 48 hr after treatment, and maintains its activity 24 hr after treatment. Methyl green staining indicates that DNase activity is accompanied by a decrease in DNA content of the tumor cells. Examination of 1-µm sections of the vitamin-treated tumors indicates that vitamin-induced DNase reactivation and the subsequent degradation of DNA are sufficient to induce tumor cell death. Although both necrotic and apoptotic cell death are observed in the tumor, the primary form of vitamin-induced tumor cell death is autoschizis, a novel type of necrosis characterized by exaggerated membrane damage and the progressive loss of cytoplasm through a series of self-excisions. These self-excisions typically continue until the perikaryon consists of an apparently intact nucleus surrounded by a thin rim of cytoplasm containing damaged organelles (
In vivo studies designed to determine the effect of vitamin administration on the lifespan of nude mice demonstrated that mice receiving both oral and IP vitamin lived significantly longer (p<0.01) than control mice. The results of additional in vivo studies, designed to determine the effect of vitamin administration on the growth of solid tumors in nude mice, demonstrated that administration of clinically attainable doses of oral vitamins ad libitum in drinking water could significantly reduce the growth rate of solid tumors in nude mice (p<0.05). Finally, nude mice receiving the vitamin combination did not exhibit any significant bone marrow toxicity, changes in organ weight, or pathological changes in these organs (
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Acknowledgments |
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Supported by grants from the American Institute for Cancer Research, 97B048, the Summa Health System Foundation of Akron, Ohio, and the HessRoth Kaminski & Maxon Foundation of Erie, Pennsylvania.
Received for publication April 28, 2000; accepted August 5, 2000.
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