Optimal conditions for immunohistochemical determination of the in vitro DNA synthesis labelling index with bromodeoxyuridine in head and neck cancer.

The in vitro DNA synthesis labelling index was assessed immunohistochemically in 24 freshly obtained specimens of head and neck cancer using bromodeoxyuridine (BrdUrd) as the DNA precursor to determine the influence of BrdUrd concentration on labelling index (LI). Initially, tumour fragments were incubated in varying concentrations of BrdUrd from 2 to 100 microM for 2 h, and BrdUrd was detected with an anti-BrdUrd monoclonal antibody using immunoperoxidase labelling. There was a dose-response gradient with mean LI varying from 1.6% at 2 microM BrdUrd to 8.8% at 100 microM. The concentration-response gradient best fit a quadratic model when LI was plotted against log BrdUrd concentration (r = 0.65, P less than 0.0001). Eleven additional tumours were then studied to determine whether LI increased for BrdUrd concentrations above 100 microM. The mean LI at 125 microM and at 150 microM in these 11 tumours did not differ from the value at 100 microM, suggesting a plateau at this level. The gradient effect accounted for 17% of the variance in LI, while 60% of the variance was explained by between tumour differences. Within individual tumours, three response patterns were observed: (i) LI rose at a constant rate to the highest concentration tested (n = 8), (ii) the LI plateaued or declined at high BrdUrd concentrations (n = 6); and (iii) there was a biphasic slope slope in which the rate of rise in the LI increased at the higher BrdUrd concentrations (n = 2). The data show that BrdUrd concentration is an important variable in the immunohistochemical assessment of the in vitro LI in head and neck cancer.

The influence of tumor stage and metastasis on the biodistribution of gallium-67 citrate in the murine Lewis lung carcinoma model.

The biodistribution of i.v. administered [67Ga]citrate was investigated in the Lewis lung carcinoma/male B6D2F1 mouse model. Tumors were implanted intramuscularly (10(5) cells or 10(6) cells in suspension) into the thigh, or subcutaneously (10(7) cells or 2 mm3 fragments) into the tail of recipient mice. Intramuscular tumors were allowed to grow for 16, 24, or 33 days; tail tumors developed for 2 wk (fragment implants) or 3 wk (10(7) cells in suspension) after which the primary tumor was amputated along with adjacent fragments of the tail tissue. Gamma camera scintigraphy and dissection/radiometric biodistribution studies indicated that: (a) tumors and metastases took up 5-6% of the injected dose/g except when large necrotic areas were present in the primary tumor; (b) blood levels of 67Ga increased in all tumor-bearing animals, with up to tenfold increases in the i.m. tumor model at later stages of the growth; (c) hepatic uptake increased as a function of tumor size/age, and (d) all tissue:blood ratios declined as the neoplastic tissues progressed. The results are discussed with respect to tumor progression and metastatic disease.

Influence of misonidazole, SR-2508, RSU-1069 and WR-2721 on spontaneous metastases in C57BL mice.

The effect of treatments with the hypoxic cell radiosensitizers misonidazole (MISO), SR-2508, RSU-1069, and with the radioprotector WR-2721 on spontaneously disseminated Lewis lung carcinoma (LLC) and B16 melanoma (B16M) was investigated. Tumors were implanted into the tails of C57BL mice and were surgically removed after reaching volumes of approximately 40 mm3. This technique results in the induction of metastatic disease in lungs and at other anatomical sites and allows the independent treatment of disseminated tumor cells. The radiosensitizers and the radioprotector were administered 24 hr after surgery and animals were killed 14 and 30 days after removal of LLC and B16M, respectively. The time to death from spontaneous metastases was also measured. Both single treatments with large doses of MISO (1.0 g/kg) and fractionated therapy with smaller doses (0.5 g/kg on 5 consecutive days) promoted the formation of metastases to the lungs, lymph nodes and other organs. The survival times of MISO treated animals did not differ from control animals but manifestation of metastatic disease in the lungs and other organs occurred at earlier times. Administration of equitoxic doses of SR-2508 (3.0 g/kg) and RSU-1069 (0.1 g/kg) also promoted metastases formation. Mice treated with these radiosensitizers developed more metastases in the lungs and at other sites. Treatment with a single dose of WR-2721 (0.4 g/kg) promoted lung metastases but exerted a suppressive effect on lymph node tumors. When the radioprotector was given in fractionated schedules in three different doses (0.05 g/kg, 0.1 g/kg and 0.2 g/kg for 10 consecutive days) a slight enhancement of lung metastases and suppression of extrapulmonary metastases was observed.

The effectiveness of cis-platinum, cyclophosphamide and melphalan in treating disseminated tumor cells in mice.

Both B16 melanoma and Lewis lung carcinoma growing in C57/Bl mice spontaneously metastasize to the lungs and other organs. When the tumors are grown in the mouse tail to a specific volume and amputated, the spontaneously disseminated tumor cells can then be independently treated. The effects of a single dose of cyclophosphamide (200 mg/kg), cis-platinum (6 mg/kg) and melphalan (10 mg/kg) on the appearance of pulmonary and other metastases were measured. The cis-platinum treatment was shown to reduce the number and incidence of metastases of both tumors at various times after treatment. The antimetastatic effectiveness of cis-platinum against these two tumors was increased when 2.4 mg/kg was administered each day for five consecutive days after amputation of the primary. Cyclophosphamide, when administered at two-thirds maximum tolerated dose, had a small promoting effect on the number and incidence of pulmonary metastases of Lewis lung carcinoma, whereas, applied in the same dose, it had efficacy in the treatment of disseminated B16 melanoma and inhibited appearance of both pulmonary and lymph-node metastases. When melphalan was administered in single- and multiple-dose regimens, the number and incidence of metastases of both tumors increased at various times after primary tumor amputation. These data suggest that melphalan can promote the growth of disseminated tumor cells in both the lungs and other sites and that some systemic chemotherapies may result in promotion instead of suppression of metastatic disease.

The treatment of animal tumours and their metastases with 4-hydroxyanisole.

4-Hydroxyanisole (4-OHA) was administered to C57Bl/10J mice in which B16 melanoma or Lewis lung carcinoma had been implanted s.c. or i.m. The drug had the largest antitumour effect against B16 melanoma growing s.c. and a smaller antitumour effect against B16 melanoma or Lewis lung carcinoma growing i.m. In the treatment regimens where drug was administered only after tumour implantation, a significant reduction in number of spontaneous metastases and their incidence was observed. Again, the largest antimetastatic effect was observed for s.c. B16 melanoma with smaller effects observed for i.m. B16 melanoma or Lewis lung carcinoma. Experiments in which 4-OHA treatment was initiated after amputation of the primary tumour implanted in the tail confirmed that 4-OHA did have antitumour activity against disseminated tumour cells. The drug regimens studied to date produced significant delays in the appearance of spontaneous metastases in the lungs and significant increases in the life spans of the treated animals.

Influence of aspirin and/or anabolic steroid (nandrolone decanoate) on experimental metastasis formation.

The influence on transpulmonary passage by an inhibitor of platelet aggregation, aspirin, and/or anabolic steroid, (Nandrolone decanoate), was investigated in syngeneic CBA mice. An increase of extrapulmonary metastases was used as a measure of increased redistribution of tumor cells from lungs to other organs. Aspirin alone did not reduce metastasis formation in lungs and did not significantly increase transpulmonary passage of tumor cells. The anabolic steroid increased the metastasis crop only in the lungs. However, treatment with the two compounds together increased the metastases in lungs and extrapulmonary organs. The results disclosed that in this system inhibition of platelet aggregation did not reduce metastasis formation but in combination with another drug, an anabolic steroid, it increased the number of experimental metastases.

Influence of some synthetic antioxidants on the growth and metastases formation of Lewis lung carcinoma and amelanotic B16 melanoma in C57BL mice.

Synthetic antioxidants: EPO (p-ethoxyphenol), PHP (p-hydroxypropiophenone), and DPPD (N,N'-diphenyl-p-phenylenediamine) dissolved in dimethylsulfoxide were administered subcutaneously to mice after subcutaneous implantation of Lewis lung carcinoma or amelanotic B16 melanoma into the tails. The doses of antioxidants used were: 50 mg/kg, 100 mg/kg, and 200 mg/kg per day, respectively. The animals received five injections a week during two weeks. The tails with Lewis lung carcinoma were amputated after 16 days and with amelanotic melanoma after 20 days; tumors weight was measured. 25 days after Lewis lung carcinoma and 42 days after B16 melanoma implantation mice were sacrificed, lungs weighed and incidence of pulmonary and extrapulmonary metastases were estimated. There was a significant increase of weight of amelanotic melanoma in animals treated with EOP or PHP (p less than or equal to 0.05). No influence of antioxidants on the metastases incidence of B16 melanoma was found. Antioxidants used did not influence the Lewis lung carcinoma weight, whereas EOP or PHP increased the incidence of extrapulmonary metastases (p = 0.05, and p = 0.1, respectively).

Activity of some lysosomal hydrolases in the homogenates of transplantable melanotic and amelanotic melanoma in golden hamster (Mesocricetus auratus, Waterhouse).

Activity of arylsulphatase, beta-glucuronidase, cathepsin D, and acid phosphatase in the homogenates of melanotic and amelanotic melanoma was determined. The activity of these enzymes is higher in melanotic than in amelanotic melanoma. Respective values for melanotic and amelanotic tumours are: arylsulphatase 10,78 +/- 3,20, and 1,45 +/- 0,66 micron 4-nitrocatechole/mg protein/hr; beta-glucuronidase 11,10 +/- 1,40, and 9,98 +/- 1,35 micron phenolphthalein/mg protein/hr; cathepsin D 4,24 +/- 1,37, and 3,26 +/- 0,73 micron tyrosine/mg protein/hr; acid phosphatase 230 +/- 22, and 180 +/- 25 micron p-nitrophenol/mg protein/hr. These differences are statistically significant. The increased activity of the lysosomal enzymes in melantoic melanoma probably depends on the occurrence of an higher number of lysosomes in tissues containing melanins.

The effect of trasylol (Trascolan "Polfa") and p-aminomethylbenzoic acid (Pamba "Germed") on the experimental metastases of transplantable melanotic melanoma in golden hamster (Mesocricetus auratus, Waterhouse)

The influence of two proteinase inhibitors -- Trasylol (Trascolan "Polfa") and PAMBA ("Germed") -- on experimental metastases of melanotic melanoma has been studied in golden hamsters. Animals were injected intravenously with tumour cells (5x10(5) or 10(6) in 0.2 ml of saline). They obtained protease inhibitor intraperitoneally one hour prior, or one hour after melanoma cells injection. One group of hamsters were also inoculated with tumour cells preincubated 10 min. in saline containing Trasylol or PAMBA. A diminished number of experimental metastases to the lungs in each group has been observed.