Oncology 32: 47-51 (1975)
Mitogenic Stimulation of Lymphocytes in Cancer Patients M.M. Reddy, K.O. Goh and C. Poulter University of Rochester and Monroe Community Hospital, Rochester, N.Y.
Key Words. Lymphocyte response • Cancer • Irradiation • Mitotic rate Abstract. Peripheral blood lymphocytes obtained from 16 controls, 16 untreated can cer patients and 15 cancer patients treated with irradiation were cultured with phytohemag glutinin (PHA) and pokeweed mitogen (PWM). The cultures were terminated on 3 and 7 days of incubation, and mitotic rates were obtained. The mitotic rates of untreated cancer patients for both mitogens did not differ significantly from those of controls. However, the mitotic rate obtained for 3-day PHA culture of the radiation-treated cancer patients was significantly different from controls, whereas the 7-day mitotic rate stimulated by PWM was significantly lower than the untreated cancer patients and controls.
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 2:05:17 AM
Some plant substances can stimulate lymphocytes to divide in vitro. It appears that phytohemagglutinin (PHA) primarily stimulates thymus-dependent (T) lymphocytes, and pokeweed mitogen (PWM) stimulates both T and B (bonemarrow-derived) lymphocytes (7). The stimulation of lymphocytes by these substances probably involves mechanisms similar to the immunological process. The reports regarding the in vitro lymphocyte response to PHA from cancer patients are conflicting. Some reports (1, 15) indicated a decreased response of lymphocytes to PHA while others (6, 10) indicated no impairment of response. However, a decrease of PHA response was observed in cancer patients treated with radiation (2, 13). The majority of these studies were limited to 3-day PHA-stimulated cultures. Here we report our observations of the relative lym phocyte response to PHA and PWM, as measured by the mitotic rates at 3- and 7-day cultures from cancer patients who were or were not treated with radiation.
Reddy /Goh/Pouher
48
Materials and Methods 16 healthy individuals, aged between 20 and 73, were used as controls. The untreated cancer patients included: 5 with cancer of the lung, 2 each with cancer of the breast, prostate and stomach, and 1 each with cervix, endometrium, esophagus, lymphoma and mycosis fungoides. Their ages ranged from 25 to 86 years. The radiation-treated cancer patients included: 5, pharynx; 3, reticulum cell sarcoma; 2, cancer of unknown origin, and 1 each with cancer of the breast, lymphosarcoma, urinary bladder, tongue and multiple my eloma. Their ages ranged from 50 to 82 years. All blood samples were coded. Lymphocytes were separated with the method described (11). Briefly, 20 ml of hepa rinized peripheral blood were mixed with 3 ml of 6 % dextran and left for 1 h while the erythrocytes sedimented. The phagocytic cells were removed by passing the white blood cell rich plasma through fiberglass columns. The resulting lymphocytes (95-100% ) were washed three times with Hank’s balanced salt solution (HBSS). Two cultures were made from each blood specimen. Each culture contained 4 -6 X 106 lymphocytes suspended in 1.0 ml HBSS, 7 ml TC 199 tissue culture medium (Bioquest, Cockeysville, Md.) and 2 ml of fetal calf serum (Grand Island Biological Co., N.Y.). One of the cultures were stimulated with 0.15 ml of PHA-M (Difco, Detroit, Mich.) and the other with 0.15 ml of PWM (Grand Island Biological Co., N.Y.). The cultures were incubated at 37 °C. Half of the culture was terminated 3 days and the other half, 7 days after incubation. During the last hour of incubation, the cells were exposed to 20 jag colcemid/ml medium. The cells were then exposed to hypotic (0.075 M KC1) solution to swell the cells, then fixed in freshly prepared aceto-alcohol (1:3). Slides were made by the air-dry method and stained with aceto-orcein. Mitotic rate was defined as the number of mitoses per 100 cells. The method used to analyze was described previously (4). At least 3,000 cells from two slides of each culture were counted at magnification (X 450) to compute the mitotic rate.
Results
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The mean mitotic rates of the lymphocyte cultures from the controls and cancer patients, either treated with radiation or not, are summarized in table I. The mean mitotic rate of the 3-day cultures stimulated with PHA for normal controls was 0.41 ± 0.07 (SE) and for untreated cancer patients was 0.32 ± 0.07. In the 7-day cultures, the mean mitotic rate was 0.48 ±0.12 for the controls and 0.36 ± 0.07 for the untreated cancer patients. These differences were not statisti cally significant. The mean mitotic rate was 0.21 ± 0.04 when the radiationtreated cancer patients’ lymphocytes were stimulated with PHA for 3 days as compared to 0.41 ± 0.07 in normal controls. This difference is statistically significant ( p < 0.0125). The mitotic rates of the PHA-stimulated cultures in creased after prolonged culturing in the normal controls and in the cancer patients. The mean mitotic rate was 0.07 ± 0.02 in the normal controls, 0.18 ± 0.04 in the untreated cancer patients and 0.08 ± 0.02 in the cancer patients treated with radiation when the cultures were stimulated with PWM and terminated
Lymphocyte Stimulation in Cancer
49
Table I. Effect of radiation treatment of cancer on mitotic rate at 3 days and 7 days of incubation with PHA and PWM Groups
Control (n = 16) Untreated cancer patients (n = 16) p value Cancer patients treated with radiation (n = 15) p value p value for untreated compared to treated cancer patients
PHA
PWM
3 days
7 days
3 days
0.41 ± 0.07
0.48 ± 0.12
0.07 ± 0.02 0.65 ± 0.08
0.32 ± 0.07 NS
0.36 ± 0.07 NS
0.18 ± 0.04 0.48 ± 0.08 < 0.025 NS
0.21 ± 0.04
Mitogenic Stimulation of Lymphocytes in Cancer Patients M.M. Reddy, K.O. Goh and C. Poulter University of Rochester and Monroe Community Hospital, Rochester, N.Y.
Key Words. Lymphocyte response • Cancer • Irradiation • Mitotic rate Abstract. Peripheral blood lymphocytes obtained from 16 controls, 16 untreated can cer patients and 15 cancer patients treated with irradiation were cultured with phytohemag glutinin (PHA) and pokeweed mitogen (PWM). The cultures were terminated on 3 and 7 days of incubation, and mitotic rates were obtained. The mitotic rates of untreated cancer patients for both mitogens did not differ significantly from those of controls. However, the mitotic rate obtained for 3-day PHA culture of the radiation-treated cancer patients was significantly different from controls, whereas the 7-day mitotic rate stimulated by PWM was significantly lower than the untreated cancer patients and controls.
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 2:05:17 AM
Some plant substances can stimulate lymphocytes to divide in vitro. It appears that phytohemagglutinin (PHA) primarily stimulates thymus-dependent (T) lymphocytes, and pokeweed mitogen (PWM) stimulates both T and B (bonemarrow-derived) lymphocytes (7). The stimulation of lymphocytes by these substances probably involves mechanisms similar to the immunological process. The reports regarding the in vitro lymphocyte response to PHA from cancer patients are conflicting. Some reports (1, 15) indicated a decreased response of lymphocytes to PHA while others (6, 10) indicated no impairment of response. However, a decrease of PHA response was observed in cancer patients treated with radiation (2, 13). The majority of these studies were limited to 3-day PHA-stimulated cultures. Here we report our observations of the relative lym phocyte response to PHA and PWM, as measured by the mitotic rates at 3- and 7-day cultures from cancer patients who were or were not treated with radiation.
Reddy /Goh/Pouher
48
Materials and Methods 16 healthy individuals, aged between 20 and 73, were used as controls. The untreated cancer patients included: 5 with cancer of the lung, 2 each with cancer of the breast, prostate and stomach, and 1 each with cervix, endometrium, esophagus, lymphoma and mycosis fungoides. Their ages ranged from 25 to 86 years. The radiation-treated cancer patients included: 5, pharynx; 3, reticulum cell sarcoma; 2, cancer of unknown origin, and 1 each with cancer of the breast, lymphosarcoma, urinary bladder, tongue and multiple my eloma. Their ages ranged from 50 to 82 years. All blood samples were coded. Lymphocytes were separated with the method described (11). Briefly, 20 ml of hepa rinized peripheral blood were mixed with 3 ml of 6 % dextran and left for 1 h while the erythrocytes sedimented. The phagocytic cells were removed by passing the white blood cell rich plasma through fiberglass columns. The resulting lymphocytes (95-100% ) were washed three times with Hank’s balanced salt solution (HBSS). Two cultures were made from each blood specimen. Each culture contained 4 -6 X 106 lymphocytes suspended in 1.0 ml HBSS, 7 ml TC 199 tissue culture medium (Bioquest, Cockeysville, Md.) and 2 ml of fetal calf serum (Grand Island Biological Co., N.Y.). One of the cultures were stimulated with 0.15 ml of PHA-M (Difco, Detroit, Mich.) and the other with 0.15 ml of PWM (Grand Island Biological Co., N.Y.). The cultures were incubated at 37 °C. Half of the culture was terminated 3 days and the other half, 7 days after incubation. During the last hour of incubation, the cells were exposed to 20 jag colcemid/ml medium. The cells were then exposed to hypotic (0.075 M KC1) solution to swell the cells, then fixed in freshly prepared aceto-alcohol (1:3). Slides were made by the air-dry method and stained with aceto-orcein. Mitotic rate was defined as the number of mitoses per 100 cells. The method used to analyze was described previously (4). At least 3,000 cells from two slides of each culture were counted at magnification (X 450) to compute the mitotic rate.
Results
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 2:05:17 AM
The mean mitotic rates of the lymphocyte cultures from the controls and cancer patients, either treated with radiation or not, are summarized in table I. The mean mitotic rate of the 3-day cultures stimulated with PHA for normal controls was 0.41 ± 0.07 (SE) and for untreated cancer patients was 0.32 ± 0.07. In the 7-day cultures, the mean mitotic rate was 0.48 ±0.12 for the controls and 0.36 ± 0.07 for the untreated cancer patients. These differences were not statisti cally significant. The mean mitotic rate was 0.21 ± 0.04 when the radiationtreated cancer patients’ lymphocytes were stimulated with PHA for 3 days as compared to 0.41 ± 0.07 in normal controls. This difference is statistically significant ( p < 0.0125). The mitotic rates of the PHA-stimulated cultures in creased after prolonged culturing in the normal controls and in the cancer patients. The mean mitotic rate was 0.07 ± 0.02 in the normal controls, 0.18 ± 0.04 in the untreated cancer patients and 0.08 ± 0.02 in the cancer patients treated with radiation when the cultures were stimulated with PWM and terminated
Lymphocyte Stimulation in Cancer
49
Table I. Effect of radiation treatment of cancer on mitotic rate at 3 days and 7 days of incubation with PHA and PWM Groups
Control (n = 16) Untreated cancer patients (n = 16) p value Cancer patients treated with radiation (n = 15) p value p value for untreated compared to treated cancer patients
PHA
PWM
3 days
7 days
3 days
0.41 ± 0.07
0.48 ± 0.12
0.07 ± 0.02 0.65 ± 0.08
0.32 ± 0.07 NS
0.36 ± 0.07 NS
0.18 ± 0.04 0.48 ± 0.08 < 0.025 NS
0.21 ± 0.04
