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Here we present a statistically rigorous approach to quantifying microarray expression data that allows the relative effects of multiple classes of treatment to be compared and incorporates analytical methods that are common to quantitative genetics. From the magnitude of gene effects and contributions of variance components, we find that gene expression in adult flies is affected most strongly by sex, less so by genotype and only weakly by age (for 1- and 6-wk flies); in addition, sex x genotype interactions may be present for as much as 10% of the Drosophila transcriptome. This interpretation is compromised to some extent by statistical issues relating to power and experimental design. Nevertheless, we show that changes in expression as small as 1.2-fold can be highly significant. Genotypic contributions to transcriptional variance may be of a similar magnitude to those relating to some quantitative phenotypes and should be considered when assessing the significance of experimental treatments.

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The delayed responses of C3H mice which had been pretreated with various single-dose and two-dose fractionated Adriamycin/X-irradiation protocols were evaluated by stressing the 120-day survivors with either whole-abdomen X-irradiation (LD50/7 assay) or whole-body X-irradiation (crypt colony survival). Pretreatment with Adriamycin alone was as toxic as Adriamycin plus X-irradiation for the animals stressed at 120 days (LD50/7 assay). There was no induced cellular radioresistance (D0) and no apparent increase in crypt size as indicated indirectly by the 10-clone dose at 120 days after completion of treatment. The increased lethality of the X-irradiation-stressed 120-day survivors was most likely a primary gastrointestinal response with little or no contribution from either bone marrow or kidney toxicity. The effect was apparently due to a persistent Adriamycin-induced antiproliferative response at the cellular level but the molecular mechanisms are unknown. Such data suggest caution to our clinical colleagues. Cancer patients treated with high doses of Adriamycin, independent of concomitant X-irradiation, will most likely be moderately to severely compromised in their ability to respond to a stress which requires cellular proliferation, and, based on the murine data, this effect is persistent if, indeed, not permanent.

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The problems encountered in studying the heterogeneity of cells in solid tumors is reviewed with emphasis on the role of various analytical cytometric assays for studying both the biology and the dynamics and proliferating, quiescent and dead malignant cells in vitro and in vivo. Due to advances in cytometric technology, many interesting in vitro studies on tumor cells heterogeneity have been and will be conducted over the next several years. For example, the acidic acridine orange staining of HeLa cells in suspension culture does readily discriminate between proliferating and quiescent cells. Some of these assays have been and others will be extended to in vivo studies. However, it is obvious that either the current analytical cytometric techniques must be modified and refined to permit better resolution for the complex situation in vivo or other new analytical cytometric techniques will have to be developed before many interesting studies on tumor cell heterogeneity in vivo can be addressed with reasonable efficiency.

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The effects of a single intraperitoneal injection of adriamycin (10 mg/kg) on a fast-growing C3H mouse mammary tumor (S102F) have been analyzed volumetrically, biochemically, autoradiographically and flow cytometrically. Mathematical simulation of the data was also used to aid in the interpretation of the recovery kinetics. This dose of adriamycin did not induce regression in tumor volume but did inhibit the growth rate for 4-5 days. 3H-TdR incorporation was gradually inhibited to reach a low of 20% of control at 24 and 36 hr and then recovered back to control by 96 hr after adriamycin treatment. The flow cytometric analysis also showed a marked reduction in the relative fraction of cells in the S-phase with a minimum of 23% of control at 72 hr; however, in contrast to the 3H-TdR incorporation data, the fraction of cells in the S-phase was only at 39% of control at 96 hr after the adriamycin injection. Since the 3H-TdR incorporation data disagreed with the flow cytometry data, autoradiographic analysis was also done at selected times after the adriamycin injections, and qualitatively, this analysis confirms the flow cytometry data in that the labeling index was 29% of control at 96 hr after adriamycin. The mitotic index also dropped from 8 to 1%, respectively, for controls and at 96 hr posttreatment. The degenerate index was about 1% in control tumors and no increase was observed in treated tumors. Adriamycin-induced cell-cycle delay occurs predominately in G1 and G2 but there is also an apparent minor delay in the transit across the S-phase and some apparent cytotoxicity in G2 and/or M. The long delay in volumetric growth appears to be due to the extended cell-cycle delay rather than extensive cell killing.

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Previous data on the effects of a single dose of hydroxyurea on C3H mouse duodenum and mammary tumors from a fast growing line (S102F) were used to predict times that may be optimal (i.e., minimize killing of the duodenal S-phase cells while enhancing the killing of tumor S-phase cells) for the administration of subsequent doses of hydroxyurea. These predicted protocols were tested by giving tumor-bearing mice injections of 2 doses at 24 hr intervals. A preliminary in vivo tumor treatment experiment was also done wherein multiple doses (up to 10) were given either at 12, 20, or 24 hr intervals with the mouse survival, body weights, and tumor volumes being recorded daily. The data show that partial cell synchronization was achieved in both tissues and the initial knetics of the surviving cells was essentially the same after a single dose, 2 doses, or 4 doses of hydoxyurea. Also, the different intervals between the 2 doses did not affect the timing of the initital peaks of DNA synthesis in partially synchronized cells; however, the height of the peaks was affected The results demonstrate that kinetic data can be useful for predicting optimal intervals for 2-dose regimes and probably multiple-dose regimes involving a single cell-cycle phase-speeific drug when applied to a mouse tumor model. However, the recovery phenomena in the respective tissues are extremely complicated and more animal tumor data need to be collected before one can make adquate use of cell-synchronizing agents and perturbed cellular kinetic data for routine clinical chemotherapy or combined modality therapy.

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