Thomas L. Avery

Site-Selective Cyclic AMP Analogs as New Biological Tools in Growth Control, Differentiation, and Proto-oncogene Regulation

The physiologic role of cyclic adenosine monophosphate (cAMP) in the growth control of a spectrum of human cancer lines, including leukemic lines, and v-rasH oncogene-transformed NIH/3T3 cells is demonstrated by the use of site-selective cAMP analogs. These cAMP analogs, which can select either of the two known cAMP binding sites of the cAMP receptor protein, induce potent growth inhibition, phenotypic change, and differentiation (leukemic cells) of cancer cells at micromolar concentrations with no sign of cytotoxicity. The growth inhibition parallels selective modulation of cAMP-dependent protein kinase isozymes, type I versus type II, and suppression of cellular proto-oncogene expression. Site-selective cAMP analogs thus provide new biological tools for investigating cell proliferation and differentiation and also for the improved management of human cancers.

Response of L1210 to combinations of cytosine arabinoside and VM-26 or VP16-213

Abstract Each of two promising new oncolytic drugs, 4′demethylepipodophyllotoxin 9 -( 4,6 - O -2-thenylidene-β- d -glucopyranoside) (VM- 26 ) and 4′-demethylepipodophyllotoxin 9 -( 4,6 - O -ethylidene-β- d -glucopyranoside) (VP 16–213 ), was administered simultaneously with cytosine arabinoside (Ara-C) to BDF 1 mice inoculated i.p. with L 1210 ascites tumor cells. The therapeutic effectiveness of these drug combinations and the interdrug relationships contributing to the observed responses were evaluated. Sixteen combinations of VM- 26 and Ara-C produced cures in eight treatment groups and, for mice that died with tumor, survival was extended 6·42 to 20·39 days (88–280%) over controls. Similarly, combinations of VP 16–213 and Ara-C produced cures in 10 of 16 treatment groups and mice that died with tumor lived 7·12–21·72 days (98–298%) longer than controls. None of the drugs given singly on the same schedule cured mice or extended mean survival beyond controls by more than 9·62 days (132%) . Responses obtained with combinations of VM- 26 and Ara-C were qualitatively similar to those obtained with combinations of VP 16–213 and Ara-C. Simultaneous administration of Ara-C, while extending survival, neither antagonized nor potentiated the response to VM- 26 or VP 16–213 . Conversely, each podophyllum potentiated the response to Ara-C. Each 100 -mg/kg increase in Ara-C extended survival by 0·6 days when the drug was administered alone, by 1·3 days when given with VM- 26 , and by 1·4 days when given with VP 16–213 . Dosage interactions between Ara-C and either podophyllum were not statistically significant. Thus, within the employed limits of dosage, small quantities of podophyllum were as effective as larger quantities in potentiating the response to Ara-C. When comparably effective levels of Ara-C and VM- 26 or VP 16–213 were combined, variation in Ara-C dosage accounted for approximately two-thirds of the treatment-related variation in survival. Inasmuch as survival increased as a function of increasing Ara-C dosage, this observation emphasizes the importance of high Ara-C dosage to the success of treatment with combinations of Ara-C and the epipodophyllotoxins, VM- 26 and VP 16–213 .

VM‐26 and cytosine arabinoside combination chemotherapy for initial induction failures in childhood lymphocytic leukemia

Combination chemotherapy with VM‐26 and ara‐C was given to 14 children with acute lymphocytic leukemia who had not responded to initial treatment with prednisone, vincristine, daunomycin, and asparaginase. Nine of these patients had also received ara‐C. At diagnosis, five children were classified as having standard prognostic features and nine as being at high risk for treatment failure. The drug combination was administered by vein twice a week for four weeks at dosages of 165 mg/m2 for VM‐26 and 300 mg/m2 for ara‐C. Nine complete remissions, five in patients with high‐risk leukemia, were induced with acceptable toxicity; all 9 subsequently were given continuation therapy with oral mercaptopurine and methotrexate. Four of the 9 patients have relapsed at 2–21 months. All treatment was stopped in 2 patients after 30 months of complete remission. Combinations of VM‐26 and ara‐C represent an alternative remission induction treatment for patients who fail to attain initial remission with agents of established effectiveness. These agents may especially benefit patients with prognostic features indicating a high risk of treatment failure.

Combined VM‐26 and cytosine arabinoside in treatment of refractory childhood lymphocytic leukemia

On the basis of previous findings at this institution, VM‐26 and Cytosine Arabinoside (ara‐C) were used in combination to treat 33 children with refractory acute lymphocytic leukemia (ALL). Chemotherapy was given by vein twice a week for four weeks at dosages of 300 mg/m2 for ara‐C and 50, 75, 110, 165, or 200 mg/m2 for VM‐26. Ten marrow remissions (nine complete and one partial) were induced, with hypotension (2/33) and bone marrow hypoplasia (20/33) the most significant side effects observed. Therapeutic responses were obtained with each dosage of VM‐26 except 75 mg/m2; myelosuppression developed at all dosages, being most prolonged at 200 mg/m2. Ten of the 23 non‐responders did not complete their planned courses of therapy. The significance of this information is that combinations of VM‐26 and ara‐C were effective in patients who were either in late stages of their leukemia or had never achieved an initial remission. All had been previously treated with prednisone, vincristine, daunomycin and L‐asparaginase. In addition, seven of the 10 responders had previously received ara‐C in other drug combinations. The use of VM‐26 and ara‐C in combination may be warranted for newly diagnosed patients who are at high risk for treatment failure with first‐line drugs.

Dose-related synergism of cytosine arabinoside and methotrexate against murine leukemia L1210

Abstract When administered in rapid succession, cytosine arabinoside and methotrexate acted synergistically in prolonging the lives of BDF 1 mice inoculated with L 1210 ascites tumor cells. The magnitude of this synergism increased as the dosage of either or both drugs was increased. At maximum dosage ( 12 mg methotrexate per kg plus 1200 mg of cytosine arabinoside per kg), the drug combination completely eradicated tumor cells in 11 of 40 mice and extended the mean survival of the remaining 29 animals by 23·64 days ( 332% ) beyond control survival. In contrast, neither drug given singly on the same schedule cured leukemic mice nor extended their mean survival beyond controls by more than 10·18 days ( 143% ). Statistically significant contributions to survival were made by three experimental variables: (i) methotrexate dosage, (ii) cytosine arabinoside dosage, and (iii) dosage interaction between the two drugs.

Activation of the respiratory burst in murine phagocytes by certain guanine ribonucleosides modified at the 7 and 8 positions: possible involvement of a pertussis toxin-sensitive G-protein

The capacity of certain guanine ribonucleosides (modified at the 7 and/or 8 positions) to enhance the respiratory burst of murine peritoneal phagocytes was evaluated. The results show that 8-mercaptoguanosine, 8-bromoguanosine, 7-methyl-8-oxoguanosine and 7-thia-8-oxoguanosine, when injected intraperitoneally into mice, induced peritoneal phagocytes to generate reactive oxygen species as early as 1 h after injection. In vivo administration of the nucleosides induced higher levels of phagocyte activation than in vitro treatment with the same nucleosides. However, the addition of interferon alpha/beta in vitro significantly increased the magnitude of phagocyte activation by the nucleosides, suggesting an important role for cytokines/lymphokines in the nucleoside-induced phagocyte activation in vivo. Furthermore, pre-treatment of phagocytes in vitro with Bordetella pertussis toxin, before treatment with the guanosines, inhibited their capacity to induce the respiratory burst. These observations establish these low-molecular-weight compounds as interesting probes for the study of stimulus-response coupling in phagocytes.

Circadian dependence of host and tumor responses to cyclophosphamide in mice.

Abstract In a series of five experiments, 505 female BDF 1 mice were standardized for periodicity studies by exposure to alternating periods of 12 hr of light and 12 hr of darkness. The mice were inoculated intraperitoneally with 1 million L1210 murine leukemia cells and beginning approximately 24 hr later, were injected by the same route with a single dose of cyclophosphamide at 1500, 1800, 2100, 0600 or 0900 hr . At each time point, different groups of mice were treated with 300 or 360 mg of cyclophosphamide per kg of body weight. Cured mice ( 60-day survivors) as well as mice dying from drug-induced toxicity or tumor were noted for each interval of treatment. The magnitude of the effects produced by cyclophosphamide varied widely from experiment to experiment, suggesting that factors presently unidentified also influenced host responses to the drug. Nevertheless, the composite data indicated that both the therapeutic and the toxic effects of cyclophosphamide were quantitatively related to the circadian phase of drug administration. The frequency of cures was highest ( 34% ) when the drug was administered at 1800 hr and lowest ( 7% ) when given at 0900 hr . In addition, drug-induced toxicity exhibited a reverse pattern of cyclic activity in which the minimum value ( 20% ) resulted from administration at 1800 hr while the maximum number of deaths ( 52% ) occurred after treatment at 0900 hr . Due, in part, to this reciprocal relationship between cure rate and drug-induced death rate, the selective timing of cyclophosphamide administration resulted in greatly divergent modification of the risk versus benefit ratio. As the result of treatment at 0900 hr , approximately 7 mice were killed for each mouse that was cured while, after treatment at 1800 hr, 1.7 mice were cured on the average for each mouse that was killed. Thus by taking advantage of temporal fluctuations in host resistance to cyclophosphamide, enhanced therapeutic responses without equivalent increases in toxicity may be produced.

CONTINUOUS NEGATIVE CHEST‐WALL PRESSURE THERAPY FOR ASSISTING VENTILATION IN OLDER CHILDREN WITH PROGRESSIVE RESPIRATORY INSUFFICIENCY

Abstract. Continuous negative chest‐wall pressure (CNP) was used to assist ventilation in 14 children, 6 months to 14 years of age, who had progressive respiratory insufficiency caused by diffuse bilateral alveolar disease. Before the start of CNP therapy, each child had a respiratory rate>50/min, arterial oxygen tension (PaO2)<70 mmHg (FIO2≥50%), and arterial carbon dioxide tension (PaCO2)<45 mmHg. The mean intrapulmonary right‐to‐left shunt was 28.7±3.8%. Within 6 hours after therapy was started, PaO2 increased from 55.4±15.9 to 81.6±17.7 mmHg (p<0.005). This improvement was sustained and within 24 hours permitted a decrease in fractional concentration of inspired oxygen (FIO2) from 51.8±6.2 to 41.0±8.4% (p<0.001) and in respiratory rate from 78.1±23.0 to 56.4±21.3 (p<0.01). There was a concomitant decrease in intrapulmonary right‐to‐left shunt. Four of the 14 patients developed pneumothorax that was successfully decompressed. Ten patients survived.

Pheromone-Induced Changes in the Acidophil Concentration of Mouse Pituitary Glands

Pituitaries of female mice in anestrus resulting from colony housing were characterized by a 58.0-percent (acidophil content. Subsequent exposure to restrained male mice for one and two nights failed to evoke significant acidophilic degranulation and resulted in pituitary acidophil values of 57.4 and 55.1 percent respectively. Exposure to released males on the third night produced marked acidophilic degranulation resulting in a significant decline in pituitary acidophils to 38.0 percent. These findings support the view that female pheromone suppresses and male pheromone favors the secretion of follicle-stimulating hormone and indicate that luteotrophic hormone is secreted at its assigned time in the sequence of cyclic ovarian events initiated by the secretion of follicle-stimulating hormone.

Nucleoside and Nucleotide Modulation of Oncogenic Expression: A New Approach to Cancer Chemotherapy

The last 15 years have seen a virtual explosion in knowledge of cancer at the molecular level. Major efforts in the area of molecular genetics were stimulated by a study of viral oncogenes (1) and have centered around the activation of proto-oncogenes, which code for proteins that are involved in the signal transduction events that modulate normal cellular growth and differentiation. More that one hundred normal cellular proto-oncogenes are now known (2). Mechanisms of activation of proto-oncogenes to cellular oncogenes include point mutation, deletion, insertion, amplification, activation by internal rearrangement, chromosomal translocation, and promoter insertion (3). Cancer would appear to have many causes, but a common element is DNA damage resulting in aberrant gene expression which is a multi-step process (4). Chromosomal analyses of tumor cells have revealed many abnormal karyotypes with metastases as the most aberrant. Most cancers exhibit some cytogenetic defect (5).