In previous clinical trials, recombinant interleukin-2 (rIL-2) has been infused at high doses over short periods of time to generate lymphokine-activated killer (LAK) cells in vivo. These trials have been limited by severe toxicities, and the immunologic effects of rIL-2 have been transient. The present study was designed to assess the toxicity and immunologic effects of prolonged administration of low doses of rIL-2. In this phase I study, patients with advanced cancer were scheduled to receive intravenous (IV) infusion of rIL-2 without interruption for 3 months in an outpatient setting. Twenty-one patients received rIL-2 at doses ranging from 0.5 x 10(5) to 6.0 x 10(5) U/m2/d. Treatment was extremely well tolerated, and no patient experienced grade 3 or grade 4 toxicity. The lowest dose level (0.5 x 10(5) U/m2/d) did not have demonstrable immunologic activity. At doses of 1.5 x 10(5) and 4.5 x 10(5) U/m2/d, rIL-2 infusion resulted in the specific expansion of natural-killer (NK) cells (sixfold and ninefold increases, respectively, at these two dose levels) without any changes in B cells, T cells, neutrophils, or monocytes. Grade 2 toxicity was observed at the dose of 6.0 x 10(5) U/m2/d, as three patients required interruption of therapy and two patients who completed therapy developed transient hypothyroidism. In patients with increased NK cells, enhancement of non-major histocompatibility complex (MHC)-restricted cytotoxicity and increased generation of LAK cells in vitro were also demonstrated. Therapy with low-dose rIL-2 can be given safely in an uninterrupted fashion for prolonged periods of time in an outpatient setting. This results in selective expansion of NK cells in vivo with minimal toxicity. Further investigation of this schedule for immunomodulation in vivo should be pursued in phase II studies of both malignant and immunodeficient disease states.

Synthesis of avian apo very-low-density lipoprotein (apoVLDL)II is estrogen dependent and liver specific. Competence to express the apoVLDLII gene is not acquired until days 7-9 of embryogenesis and thus lags 5-6 days behind appearance of the liver primordial bud. It is not known whether the delayed ability to activate the gene is attributable to hepatic estrogen receptor profiles, or a requirement for other transcription factors not expressed at earlier stages of embryogenesis. The latter possibility is supported by developmental alterations in nuclease hypersensitivity flanking the gene that occur independently of estrogen administration. We have examined the influence of these hypersensitive regions on expression from the apoVLDLII promoter and have characterized novel protein-DNA interactions at two of them. One is located in a copy of the CR1 family of middle repetitive elements approximately 3.0 kb upstream from the start of the gene. We demonstrate by DNase I footprinting that the site contains an element which matches a predicted consensus silencer sequence. The other site contains no previously identified binding motifs. It is located between nucleotides -228 and -245 and is adjacent to an imperfect estrogen response element (ERE) that we demonstrate acts additively with a canonical ERE 30 nucleotides downstream. We have identified ubiquitous and liver-specific factors that display overlapping DNA contacts with the site. Mutation of G residues contacted by these proteins decreases hormone-inducible expression from the promoter 5- to 8-fold. Hepatic levels of the liver-enriched factor interacting with this site increase abruptly between days 7 and 9 of embryogenesis, suggesting that it may be an important determinant of the ability to express the apoVLDLII and possibly other liver-specific genes.

The proliferative responses of rat prostate and human benign prostatic hyperplasia have been followed in organ culture using [125I] iododeoxyuridine uptake to monitor DNA synthesis. In serum-free cultures, testosterone induced a marked increase in DNA synthesis (three-fold) in 4- to 6-month-old rat prostates at concentrations of 4 x 10(-9) to 4 x 10(-6) M, whereas in greater than 12-month-old rat prostates the response was less marked. Human benign prostatic hyperplasia also showed an increased uptake at similar testosterone concentrations and of a similar magnitude to the response of greater than 12-month-old rat prostates. At 10(-5) M DNA synthesis was markedly suppressed in cultures of both rat and human prostate. The proliferative response of human benign prostatic hyperplasia increases up to days 3 to 4 in culture and then declines in both control and hormone-treated groups and may represent repair processes which appear to be hormone dependent.

Effects of variations in organ culture media and methodology on the proliferative activity of rat ventral prostate and its response to testosterone were investigated quantitatively by using the incorporation of [125I]-iododeoxyuridine (125I-UdR) to monitor DNA synthesis. In serum-free medium, maximal increases in DNA synthesis occurred when testosterone was introduced during the first 48 hours of the culture. Supplementation of the medium with 5% fetal calf serum did not alter the responses in control or testosterone-treated cultures, whereas the addition of insulin (3 micrograms/ml) alone or in combination with serum promoted cell proliferation in testosterone-free cultures and enhanced the stimulatory effect of testosterone. Unlike grid-supported cultures, suspension cultures of rat ventral prostate exhibited increased proliferative activity both in the presence and absence of testosterone. Thus, variations in experimental procedure may account for differences in the proliferative activity of rat prostate between organ culture studies.

Proliferative responses of normal rat ventral prostate to testosterone were compared with that of its major metabolites, 5α‐dihydrotestosterone, androstenedione, androstanedione, and 5α‐androstane‐3β,17β‐diol in serum‐free organ culture medium using the incorporation of 5‐[125I]‐iodo‐2′‐deoxyuridine (125I‐UdR) to monitor DNA synthesis. With the exception of 5α‐androstane‐3β,17β‐diol, these androgens induced comparable increases in DNA synthesis at both 4 × 10−9 and 4 × 10−7 M, and at 4 × 10−5 M they exerted a nonspecific cytotoxic effect, resulting in marked suppression of 125I‐UdR uptake. In contrast, 5α‐androstane‐3β,17β‐diol was not stimulatory at 4 × 10−9 M, whereas at both 4 × 10−7 and 4 × 10−5 M it stimulated marginal increases in DNA synthesis and effectively maintained the histology of the tissue in a state comparable to the intact gland. Unlike 5α‐dihydrotestosterone, its epimer 5β‐dihydrotestosterone was inactive at all concentrations used. These results support the hypothesis that testosterone action in the prostate is mediated by the formation of active 5α‐reduced metabolites.