Simultaneous flow cytometric deoxyribonucleic acid and acid phosphatase analysis of benign and malignant lesions of the prostate.

Flow cytometry can differentiate benign from malignant lesions of the prostate through deoxyribonucleic acid distribution analysis. A method has been developed that permits simultaneous cytometric determination of deoxyribonucleic acid and acid phosphatase activity in the cell cycle compartments of prostatic biopsy specimens. Histograms of prostatic carcinoma reveal higher acid phosphatase activity and greater deoxyribonucleic acid content in the S and S + G2/M populations than the histograms representing benign lesions. This compartmental difference may have prognostic usefulness.

Flow cytometric DNA and 5'-nucleotide phosphodiesterase in endometrium.

One hundred endometrium specimens have been studied with flow cytometry for DNA analysis (FCDA) and a proliferative enzyme marker, 5'-nucleotide phosphodiesterase (5'-NPD). FCDA data showed that aneuploidy was present in only 5 of 40 cancer specimens. However, with corrected histograms, a higher DNA value was observed in the G2/M (6%) of all cancer compared with noncancer specimens (4%). Thus, FCDA can be a useful diagnostic aid for endometrial cancer. The determination of 5'-NPD was done with a quenching method based on the use of 5'-(5-iodo-3-indoxyl)-thymidine phosphodiester as a substrate and 4',6-diamidino-2-phenylindole for DNA. This method could qualitatively define which population of the cell cycle had a higher enzyme level and also quantitatively gave the enzyme units per cell. It was found that 12.5% of all cancer specimens had 5'-NPD activity in the G0/G1 cells and 87.5% in the S and/or G2/M cells, whereas in the noncancer specimens 5'-NPD was found in 28.5% of the G0/G1 cells and 71.5% of the specimens had 5'-NPD in the S and/or G2/M cells. Furthermore, the concentration of 5'-NPD was found to be five times higher in the G2/M cells of the cancer specimens than that in the noncancer specimens. However, in the hyperplasia specimens, the activity was only two times higher in the same cell cycle fraction than in the normal specimens. The results of this investigation provided for the first time evidence that this exonuclease activity alters in the cell cycle fractions and that a decrease in the enzyme activity in G0/G1 cells and an increase in G2/M cells may be a useful marker for neoplastic development in human endometrial cancer.

Rational selection of adjuvant chemotherapy after cytoreduction surgery for murine neuroblastoma.

Improving the prognosis of advanced neuroblastoma remains an important yet unachieved goal of pediatric oncology, a fact which may be related to an insufficient analysis of the role played by cytoreductive surgery. Utilizing strain A mice bearing C-1300 syngeneic neuroblastoma, tumor biology and host immunocompetence were studied after cytoreduction surgery and adjuvant chemotherapy. Cell kinetic analysis in the residual tumor demonstrated an increase of the proliferative fraction 18 to 42 h after operation, but the same peak proliferation was delayed in bone marrow cells to 24 to 96 h. The potential for drug distribution to the tumor after cytoreduction surgery was assessed by injecting Na251CrO4 and measuring tumor uptake. There were two significant (P less than 0.05) peaks of activity at 6 h and 3 days, suggesting local edema and neovascularity, respectively. Injection of both cell cycle specific and nonspecific adjuvant chemotherapeutic agents in a dosage of one-fourth of their 50% lethal dose at 24 or 72 h following surgical cytoreduction did not induce any antitumor activity at either injection time. However, when cyclophosphamide was given in this dose, the C-1300 tumor growth was impaired, an effect which was largely abrogated by first subjecting the tumor bearer to thymectomy and irradiation. The transfer of spleen cells from adjuvant cyclophosphamide-treated mice to tumor-inoculated normal mice significantly delayed tumor appearance when comparison was made with animals treated by operation alone, and such recipients also exhibited a more prolonged survival. These data suggest that the antitumor activity of cyclophosphamide following cytoreduction surgery of C-1300 neuroblastoma is mediated by both pharmacological and immunological mechanisms.

Use of 111In-bleomycin for combining radiotherapy and chemotherapy on glioma-bearing mice.

Mice bearing transplanted glioma received 0.9% NaCl, 0.1 mg of BLM, or 200-250 microCi of 111In-BLM (0.1 mg BLM) daily for 5 days intraperitoneally. After therapy, tumor sizes were in the order NaCl greater than BLM greater than 111In-BLM. On the 11th day after the first injection, tumor size (mm3) in the 111In-BLM group was 1,220; in the BLM group, it was 2,310 (P less than .025). After intratumor injection of a total dose of 0.1 mg of BLM/gm tumor weight, or of 1 mCi/gm tumor weight of 111In-BLM (carried by 0.1 mg of BLM/gm tumor weight), the tumor size decreased in the 111In-BLM group more than in the BLM group. On the 5th day after the 2nd dose therapy, the tumor size in the 111In-BLM group was 2,020; in the BLM group it was 4,220 (P less than .05). Host weights for these two groups were similar. The necrotic area in the tumor was much greater in the 111In-BLM group than in the BLM group. These results suggest the use for radiotherapy and chemotherapy.

A new 111In-bleomycin complex for combined radiotherapy and chemotherapy.

Six days after tumor transplantation three daily intraperitoneal doses of 0.9% NaCl, bleomycin (BLM), or a new 111In-bleomycin complex (BLMC, 15 microCi/g body weight) were administered to glioma-bearing mice. After therapy, tumors in mice treated with 111In-BLMC were smaller than those treated with BLM. Sixteen days after the first injection tumor size for 111In-BLMC-treated mice was 560 (240-1,030) mm3, 1,980 (1,400-3,290) mm3 for BLM (P less than 0.025), and 4,830 (2,580-9,180) mm3 for NaCl (0.1 less than P less than 0.2). Thirteen days after tumor transplantation glioma-bearing mice received single intratumor injection of 0.9% NaCl, BLM, or 111In-BLMC (1.5 mCi, carried by 0.5 mg BLM/g tumor weight). The average tumor size for 111In-BLMC was smaller than that for BLM by a factor of 2.5-3.7. Host weights for these two groups were similar, and morphologic abnormalities were not found in kidney or liver.

Studies on cataractogenesis in humans and in rats with alloxan-induced diabetes. I. Cation transport and sodium-potassium-dependent ATPase.

Changes in the cation balance cause hydration and initiate the process of lens opacification. Such alterations were studied in human cataractous lenses and during the development of alloxan-induced diabetic cataract in rats by biochemical and histochemical techniques. The development of alloxan-induced cataract in rats was examined in vivo which showed cortical opacities beginning after 32 days. These opacities did progress to maturity after 64 days and finally the lenses were completely opacified after 96 days of alloxan treatment. The histochemical localization of sodium-potassium-activated adenosine triphosphatase using three different methods provided information on the possible role of this enzyme in normal and cataractous lenses. In human cataractous lenses, sodium-potassium adenosine triphosphatase activity was found to be considerably decreased, whereas no activity of this enzyme was localized in human diabetic cataractous lenses. An animal model provided evidence that an apparent decrease of sodium-potassium adenosine triphosphatase may be involved in the initiation of alloxan-induced diabetic cataract in rats.

Studies on cataractogenesis in humans and in rats with alloxan-induced diabetes. II. Histochemical evaluation of lenticular enzymes.

The effect of cataractogenesis on the behavior of some enzymes involved in glucose metabolism was examined histochemically both in human lenses and in rat lenses from rats with alloxan-induced diabetes. Several modifications in the currently available techniques were made in order to localize glucose-6-phosphate dehydrogenase, aldose reductase, sorbitol dehydrogenase, hexokinase and ketohexokinase in ocular lens. Human cataractous lenses showed a precipitous drop in glucose-6-phosphate dehydrogenase activity, whereas the lenticular tissues of alloxan-treated rats showed a gradual decrease of this enzyme with the prolongation of diabetes. Aldose reductase activity increased in hypermature and senile diabetic cataracts, whereas sorbitol dehydrogenase activity decreased in these lenses. Similarly, in alloxan-diabetic rat lenses the activity of aldose reductase increased while that of sorbitol dehydrogenase decreased with the prolongation of diabetes. Attempts were made to localize hexokinase and ketohexokinase in ocular lens.

A comparative study of DAPI, DIPI, and Hoechst 33258 and 33342 as chromosomal DNA stains.

A comparison of four DNA stains considered to be AT-specific with chromosomes from a clonal Chinese hamster cell line B14F28-C5 have been made. The flow karyotype histograms indicate that DAPI, DIPI, and Hoechst 33258 and 33342 do stain similarly in the same preparation. DAPI staining is specific and highly reproducible in this line. We, therefore, recommend this dye as a single chromosome DNA stain for high-resolution flow cytometric measurements in cytogenetics and mutation research.

5'-Nucleotide phosphodiesterase isozyme-V as a marker for liver metastases in breast cancer patients.

A 2-year prospective study testing 258 breast cancer patients for the presence of 5'-nucleotide phosphodiesterase isozyme-V (5'-NPD-V) as a marker for liver metastases was undertaken. Despite the difficulty associated with such a study in obtaining data from biopsy (only 12 patients) and autopsy (only 18 patients), the 5'-NPD-V test correctly predicted confirmed liver metastases in 39 of 41 patients (95%). Of this group, 25 patients had abnormal liver scans at the time the 5'-NPD-V test was positive (64%). In 11 of 39 patients 5'-NPD-V was found positive before liver scans. In the majority of patients, 5'-NPD-V fluctuated between positive and negative during follow-up. While such transiently positive values may be related to treatment, they should be interpreted conservatively as liver proliferation until confirmed otherwise. Throughout the 2-year study only seven patients with nonmalignant hepatic diseases had consistent positive values (2.7%). As an indicator of liver metastases 5'-NPD-V is more specific than other liver function tests. It is also important to note that 14 patients received early chemotherapy because attention was directed toward the diagnosis of liver metastases by this test.

A new tumor imaging agent--111In-bleomycin complex. Comparison with 67Ga-citrate and 57Co-bleomycin in tumor-bearing animals.

We have found a new 111In-bleomycin complex (BLMC), which has high affinity to tumor, does not bind to transferrin and is stable in vivo. Distribution in animals bearing glioma, hepatoma, or mammary adenocarcinoma at 48 hours showed: the ratios of tumor to blood, brain, heart, lung, liver, pancreas, stomach, and femur were 1.4-22.4 times as high for 111In-BLMC as for 67Ga-citrate. In mammary adenocarcinoma, 111In-BLMC bound more to viable and 57Co-Bleomycin (BLM) more to necrotic tumor. In viable tumor, the concentration of 111In-BLMC was similar to that of 57Co-BLM. The ratios of tumor to stomach and pancreas were higher, to blood, brain, muscle, heart, and femur were lower for 111In-BLMC than those for 57Co-BLM. The ratios of tumor to lung, liver, spleen, skin, and kidney were similar for the two compounds. Tumors were imaged more distinctly with the new 111In-BLMC and 57Co-BLM than with 67Ga-citrate. 111In-BLMC is promising for tumor imaging.

Flow cytometric DNA analysis as a diagnostic aid for cervical condyloma and cancer.

Flow cytometric DNA analysis data (FCDA) were obtained from 324 samples provided through the Gynecology-Oncology Clinic. These samples consisted of 294 combined endoectocervical and vaginal smears and 30 peritoneal washings. Using a conventional scheme for G0/G1, S + G2/M and the coefficient of variation with computer correction for the cell-cycle kinetics, it was possible to assign a diagnostic Class I, II, III or V similar to that used by the Cytology Laboratory. These data were then compared with the histopathologic and colposcopic diagnoses. The correlation between FCDA and cytologic results were essentially similar to the previous data obtained from only endocervical sampling. The most interesting finding in this study was the recognition of an FCDA pattern showing a higher DNA content in the G0/G1 and the early S regions in 70 of 94 (74.5%) of samples from patients with condyloma acuminata. All condyloma samples were diagnosed either by cytologic, histopathologic, or colposcopic examination, or a combination of two or three. All biopsy specimens were then reviewed by one pathologist to verify any discrepancies. The relationship of this pattern to the viral etiology of this disease is discussed with the three methods of diagnosis and electron microscopic observations. It is suggested that, based on this study. FCDA analysis of pap smears may also be useful in determining the presence of condyloma in a gynecology clinic. The potential value of FCDA analysis from peritoneal washings for the diagnosis of gynecologic cancer can not be ascertained in this preliminary investigation because of insufficient samples.

A new 111In-bleomycin complex for tumor imaging: preparation, stability, and distribution in glioma-bearing mice.

A new 111In-bleomycin complex (111In-BLMC) is here reported. Its radiochemical purity was 99% by thin-layer chromatography (TLC) (Rf 0.65) and in 5% agarose gel electrophoresis in 0.02 M NaHCO3 it migrated toward the anode. Autoradiographs of TLC and gel electrophoresis plates showed no change on storage for 3 weeks. Urine and plasma from untreated or glioma-bearing mice after injection of 111In-BLMC were analyzed by TLC and gel electrophoresis. Results indicated stability in vivo, nonbinding to transferrin, affinity to viable tumor, and excretion faster than 111In-BLM-B2, 111In-BLM, or 57Co-BLM. Tissue distributions 24 hr after injection of radiopharmaceutical showed activity ratios of tumor to blood, muscle, and brain of 13.1, 12.4, and 81.6, respectively, which were significantly higher than those for previously prepared 111In-BLM-B2 or 111In-BLM (except for brain, 0.05 less than P less than 0.1). The new 111In-BLM complex may be useful in clinical imaging and for combining radionuclide radiotherapy and chemotherapy.

Alteration of tumor cell kinetics by pulse total parenteral nutrition. Potential therapeutic implications.

Previous work has demonstrated that substrate-induced alterations of tumor metabolism can be exploited to potentiate tumor response to cycle-specific chemotherapy (methotrexate, Adriamycin [doxorubicin] ). This study was performed to investigate the biologic mechanism responsible for this phenomenon by determining the effect of short-term total parenteral nutrition (TPN) on tumor cytokinetics. Forty-two female Lewis/Wistar rats with subcutaneous mammary tumor implants (AC-33) underwent superior vena caval cannulation, and were randomized to receive either TPN or normal saline intravenously. Animals receiving TPN were killed at 2, 6, 12, 24, and 48 hours after initiating TPN; control animals given normal saline were killed at 0, 24, and 48 hours after randomization. At the time animals were killed tumor cytokinetic analysis was performed by flow cytophotometry. The percentage of tumor cells in S-phase was significantly increased in animals after only 2 hours of TPN (55.5 +/- 9.1%) compared with the control group (43.7 +/- 7.7%) (P less than 0.01). The ratio of sensitive/resistant tumor cells to S-phase-specific chemotherapy was effectively increased in animals receiving adjuvant TPN (1.31 +/- 0.43) compared with control animals (0.80 +/- 0.25) (P less than 0.015). This alteration in tumor cytokinetics provides one explanation for the enhanced tumor response to cycle-specific chemotherapy previously observed with pulse TPN administration.

Distribution and stability of [111In]bleomycin and its fractions in tumor-bearing mice.

The tissue distributions in glioma-bearing mice given injections of [111In]bleomycin (BLM) indicated that tumor concentrations and ratios of tumor to blood, muscle and brain for [111In]BLM-B2 and -A2 were higher than those for unfractionated [111In]BLM. Autoradiographs of electrophoretic gels of urine containing [111In]BLM or one of its fractions differed from those containing 111InCl3. [111In]BLM and its fractions (A2 and B2) were found to be stable in vivo. The fractions may be more useful in the clinic than [111In]BLM.

Evaluation of a one-parameter flow analysis of cervical samples for gynecology cancer screening.

A total of 246 endocervical samples were collected for Papanicolaou staining and one-parameter flow cytometric DNA analysis (FCDA) using 4',6-diamidino-2-phenylindole as a DNA stain. Typical histograms derived from FCDA analysis were designated class I, II, III, and V. Two groups of patients were studied: 135 women referred for colposcopy (COLPO), and 79 women exposed to diethylstilbestrol (DES). The remaining 32 samples were evaluated and found unsatisfactory. In the colposcopy group FCDA assigned 38 patients normal, 62 mild to moderate dysplasia, 33 moderate to severe dysplasia, and 2 carcinoma in situ and invasive carcinoma. Agreement of Pap smear and FCDA occurred in 56, 59, 72, and 100%, respectively. In the diethylstilbestrol group 57 patients were normal and 12 had mild to moderate dysplasia according to Pap diagnosis. Agreement between FCDA and Pap diagnosis were 45 and 58%, respectively. However, FCDA did not falsely assign severe dysplasia or carcinoma in situ to any DES patient. This preliminary study based on FCDA appears to clearly differentiate patients with normal Pap smears from those patients with invasive cancer.

Action of (S)-10-hydroxycamptothecin on P388 leukemia and distribution of the drug in mice.

As an inhibitor of the growth of P388 leukemia in mice, (S)-10-hydroxycamptothecin (OPT) was as potent as the parent compound camptothecin (CPT). Incorporation of thymidine into DNA was the parameter most sensitive to OPT in vitro (ED50 approximately 4 micrograms/ml), but incorporation of cytidine into RNA and of acetate into lipids was also reduced significantly in the presence of the drug. The cytofluorometric profile suggested suppression of the S and G2/M phases. The distribution of OPT in mice at 2 and 24 hours after ip injection (10 mg/kg) was essentially similar to that of CPT, with the exception of a somewhat greater concentration of CPT in the liver. In their pharmacology, OPT and CPT appear to be very similar, despite reports that the hydroxy derivative is less toxic.

Stability of 111In-bleomycin in vivo--properties compared with 57Co-bleomycin.

111Indium-bleomycin (111In-BLM) and 57Co-bleomycin (57Co-BLM) were prepared and their distributions were compared in the tissues, blood, and urine in tumor-bearing and in untreated mice and rats. Autoradiographs of electrophoresis gels showed that patterns for urine from untreated and tumor-bearing animals, collected 1-3 h or 48 h after injection of 111In-BLM were similar to those for in vitro mixtures of urine and 111In-BLM, but differed from the patterns obtained with 111InCl3 under in vivo or in vitro conditions. In rats bearing mammary adenocarcinoma, 48 h after administration of the radiopharmaceutical, the activity ratio of tumor to eleven different tissues was 1.2-4.6 times higher for injected 111In-BLM than for 111InCl3 (P less than or equal to 0.001 or P less than or equal to 0.05). Imaging with a gamma camera depicted tumors in mice more distinctly with 111In-BLM than with 111InCl3. These findings were interpreted as reflecting the stability of 111In-BLM in vivo. The tumor concentration (%dose/g) was higher for the viable area than for the necrotic area for 111In-BLM, but the reverse was true for 57Co-BLM.

Evaluation of 5'-nucleotide phosphodiesterase isozyme-V as a predictor for liver metastasis in breast cancer patients.

5'-nucleotide phosphodiesterase isozyme-V (5'-NPD-V) was evaluated in 85 biopsy proven breast cancer patients as a potential marker for early liver metastasis. It correctly predicts liver metastasis in 6/7 (85.7%) patients with abnormal radiologic liver scan and 2/2 other patients with palpable liver. Serum glutamic-oxaloacetic transaminase (SGOT), lactic dehydrogenase (LDH), alkaline phosphatase (AP) and total bilirubin (B) were also determined in 79 of these patients as routine liver function tests (LFT). Forty-one out of 79 from this group had all four markers all within normal limits. Yet of the 41 patients, 12 patients were found positive for 5'-NPD-V. Of these 12, one was found to have liver metastasis at surgery and one had abnormal liver scan. Five other patients had liver dysfunction and one had been diagnosed as an alcoholic. Four others had no evidence of either liver problems or liver metastasis, but follow-up data were lacking. This retrospective study, therefore suggests that there is a definite advantage to include the 5'-NPD-V in the liver profile studies for breast cancer patients, although a positive 5'-NPD-V may only indicate liver repair or liver regeneration. Long-term prospective studies of these tests with breast cancer patients should be worthwhile. No relation was found between 5'-NPV-V and axillary lymph node involvement or the estrogen receptor status of the excised tumor. Thus there is no evidence currently that the appearance of the 5'-NPD-V in serum is related to lymph node metastases or hormonal control.

Cell cycle and 5'-Nucleotide phosphodiesterase activities in rat liver.

The postnatal developments of liver and serum 5'-nucleotide phosphodiesterase (5'-NPD) in Fischer 344 rats were studied. The liver enzyme activities were found to correlate well with the growth of the liver as measured by the wet weight and percentages of liver cells in the G2/M phases of the cell cycle as determined by flow cytofluorometry. Therefore, this enzyme may be intimately related to the growth of the liver. Although the specific activities of the serum enzyme are three orders of magnitude less than that of the liver enzyme, very good correlation was obtained between the total serum and liver enzyme activities. This finding, therefore, suggest that the main origin of the serum enzyme is from liver. Multiple isozymes were detected in both liver and serum 5'-nucleotide phosphodiesterase after polyacrylamide gel electrophoresis.