Metabolism of a [18F]fluorine labeled progestin (21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone) in humans: a clue for future investigations.

Assessment of estrogen receptors and progesterone receptors (PR) with PET may allow the determination of the hormone responsiveness of tumors without the need for multiple biopsies, and the monitoring of the effect of hormonal therapy. In spite of the favourable characteristics of 21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone ([18F]FENP) found in preclinical studies, the compound failed to reveal the presence of PR in breast carcinomas and meningiomas. In view of the clinical significance of the PR assay in human breast cancer, it is worthwhile to explore mechanisms that are potentially involved in the inadequacy of [18F]FENP to image PR with PET. Our present study on the in vivo metabolism of [18F]FENP in humans demonstrates a rapid clearance and biotransformation of the compound. Results of incubation experiments suggest that the metabolic conversion of [18F]FENP is not restricted to the liver, but also occurs in blood cells (presumably the erythrocytes) and tumors (breast carcinomas and meningiomas). The predominant metabolite of [18F]FENP in plasma during the rapid distribution phase and in tumors is identified as 20-dihydro-[18F]FENP. The conversion of [18F]FENP to its 20 alpha- or 20 beta-hydroxy metabolite has a deleterious effect on the binding affinity for PR. Our findings do not justify a conclusion as to the extent of in vivo extrahepatic biotransformation of [18F]FENP, or its significance in the ineffectiveness of [18F]FENP as an imaging agent for PR. On the other hand, the ability of breast carcinomas and meningiomas to metabolize [18F]FENP avidly appears to preclude selective imaging of PR in these tumors during the time of a PET examination. It is imperative to evaluate the metabolic stability of a [18F]fluorine labeled progestin in an early stage of future screening procedures.

Epidermal growth factor receptor levels increase but epidermal growth factor receptor ligand levels decrease in mouse mammary tumors during progression from hormone dependence to hormone independence.

Twenty-six serially transplanted Grunder (GR) strain mouse mammary tumors were analyzed for epidermal growth factor receptor (EGFR) and EGFR-ligand levels, in addition to steroid hormone receptors (estrogen receptor, ER, progesterone receptor, PgR). In concordance with earlier studies, hormone dependent (HD) and hormone responsive (HR) tumors were found to be positive for both ER and PgR, whereas hormone independent (HI) tumors contained only 30% of the ER concentration that was found in the HD tumors. PgR was undetectable in HI tumors. HI tumors contained 2.5 to 3-fold higher EGFR levels than HD/HR tumors, an observation which shows remarkable concordance with studies on EGFR in human breast cancer. On the other hand, the level of EGFR-ligand(s) was positively associated with ER levels and was three-fold higher in HD/HR tumors than in HI tumors. The low EGFR in HD/HR tumors relative to HI tumors may be the result of downregulation by EGFR ligands produced under ER control. During progression to hormone independence this downregulation of EGFR is then abolished in absence of ER. The increase in EGFR may therefore be a secondary effect rather than a key event in the progression to hormone independence in this mouse mammary tumor model.

Phosphorylation and estradiol binding of estrogen receptor in hormone-dependent and hormone-independent GR mouse mammary tumors.

The effect of phosphorylation on the hormone-binding capacity of the estrogen receptor (ER) was investigated in hormone-dependent (HD) and hormone-independent (HI) mammary carcinomas of GR mice. Tumor cytosols were incubated with ATP under conditions previously used to study the tyrosine kinase which confers hormone binding to phosphatase-treated or in vitro-synthesized ER. The ATP-dependent increases in hormone-binding capacity of 8 out of 20 HI tumors ranged from values of 23 to 124 fmol/mg cytosol protein. The enhancement by ATP of hormone binding to ER was significantly less marked in HD and HR tumors than in HI tumors. In only 3 out of 13 HD and HR tumors was an increase ranging from 15 to 20 fmol/mg protein detected. Analysis by Scatchard plot of estradiol binding to ER showed that cytosol incubation of HI tumors with ATP markedly increased the hormone binding without any change in affinity. The data suggest that ER of HI tumors is less phosphorylated in vivo than the ER of HD/HR tumors, so that the receptor of HI tumors is more susceptible to gamma-32P-ATP phosphorylation and ATP-induced hormone binding in vitro. Western blot of ER with antiphosphotyrosine antibody showed that, in HI tumors, the large ATP-induced increase in hormone binding to ER was associated with phosphorylation on tyrosine of the receptor itself. Our findings indicate that the process of activation-inactivation of binding through tyrosine-phosphorylation/phosphotyrosine-dephosphorylation of ER observed in estrogen target tissues is altered in some HI mammary tumors.

Comparison of estrogen receptors in hormone-dependent and hormone-independent Grunder strain mouse mammary tumors.

Hormone-dependent (HD) Grunder strain mouse mammary carcinomas contain a 65-kDa estrogen receptor (ER) with minor amounts of 50- and 35-kDa components which apparently still contain the intact hormone-binding (COOH-terminal) domain. When the HD tumors lose their hormonal dependence during serial transplantation, the hormone-independent (HI) transplants show an increase in 50- and 35-kDa components relative to 65-kDa ER. In HI transplants of three of five tumor lines studied (TSl 85, 86, and 106), the 65-kDa receptor was entirely replaced by 50- and 35-kDa receptors, whereas in the two other lines (TSl 101 and 104) there usually were about equal amounts of 65- and 50-kDa ERs. No difference was found between ERs of HD and HI tumors in affinity for estradiol, steroid specificity, or immunoreactivity for the monoclonal antibody JS34/32. Estrogen stimulation of HI tumors did not increase the concentration of progesterone receptor in the tumor tissue, indicating that ER in these tumors was not functional in enhancing progesterone receptor. Incubation of 65-kDa ER with HI tumor cytosol or combined homogenization of HD and HI tumor tissue did not cause degradation of 65-kDa ER. alpha-Chymotrypsin-like protease activity generally was lower in HI than in HD tumor cytosols, indicating that the lower molecular size of ER in HI tumors cannot be attributed to the increased level of this protease activity.

Assignment of estradiol receptor gene to mouse chromosome 10.

Differences in restriction fragment lengths were detected with murine estrogen receptor cDNA (clone MOR-100) between Chinese hamster and mouse. These were used to determine the chromosomal location of the estrogen receptor in the mouse by Southern blot analysis of DNAs obtained from a panel of mouse-Chinese hamster somatic cell hybrids. The mouse estrogen receptor gene was localized on mouse chromosome 10.

Different int-1 region DNA rearrangements within different zones of a single mouse mammary tumor.

Fragments were taken from separate parts of hormone-dependent (HD) primary GR mouse mammary tumors and serially transplanted in estrone plus progesterone treated or hormonally untreated castrated mice. The transplants were examined with respect to int-1 DNA rearrangement, proviral integrations of the murine mammary tumor virus (MMTV), and estrogen and progesterone receptor content. One of the fragments (b) taken from the primary tumor of line TSI 96 produced transplants that showed int-1 rearrangement in one allele and also MMTV proviral integrations not at the int-1 gene, whereas transplants from another fragment (a) only had the normal germ-line int-1 arrangement and no extra MMTV provirus. These respective genotypes were retained when the tumors became hormonally independent during further transplantations. The results indicate that int-1 rearrangement was not present in the originally transformed cell but occurred in a HD cell during growth of the tumor. Furthermore they indicate that loss of hormonal dependence in GR mammary tumors is due to a mutational event, unrelated to int-1 rearrangement.

Hormonal regulation of mouse mammary tumor growth.

In view of reports that human breast cancer cells secrete growth factors that can replace estradiol in sustaining tumor growth [1], we have investigated whether hormone independent (HI) GR mouse mammary tumors can sustain growth of estrogen-depleted hormone dependent (HD) tumors. HD GR mammary tumor TSl 106 was grafted subcutaneously in the right flank of estrone plus progesterone treated castrated (020 X GR)F1 mice. After 2 weeks the estrone treatment was stopped and the mice received 50, 100 or 150 mg HI GR mammary tumor TSl 104 in the left flank. However, the regression of the HD tumor due to estrone depletion was not prevented or retarded by the HI grafts. In other experiments we investigated integrations of mouse mammary tumor virus (MMTV) proviral DNA in the DNA of GR mammary tumors. We could demonstrate the presence of two cell populations in tumor TSl 96, both HD but differing in MMTV DNA integration events. Our data indicate that exogenous integrations of MMTV proviruses can take place in mouse mammary tumor DNA without loss of hormone dependency of the tumors. Like in GR/Mtv-2+ mice, mammary tumor transplants differing in MMTV proviral integrations are also observed in 020/Mtv-2+ mice.

Chemotherapy and hormonal therapy of mouse mammary tumors.

Single Cisplatin (Platinol) injections i.p. at 10 mg/kg dose level (LD10) caused a marked growth inhibition of both hormone dependent and independent GR mouse mammary tumors (reduction in tumor volumes of 86 and 85%, respectively). However this treatment caused severe toxic side effects including loss of mouse body weight and deaths of some of the animals. When Cisplatin (pure) was administered s.c. in a single cholesterol pellet, no such toxic effects were observed even when Cisplatin levels up to 30 mg/kg were applied. The latter dose caused 29% reduction in tumor volume of hormone independent tumors. However this inhibitory effect was temporary, since regrowth of the hormone independent tumors started again 8 days after application of the single Cisplatin pellet. These results show that continuous Cisplatin administration in pellets s.c. is less toxic than injection i.p., but that the inhibition of mammary tumor growth is also less marked.

Changes in sensitivity to cyclophosphamide of mouse mammary tumors during serial transplantation.

The effect of the cytostatic agent cyclophosphamide on the growth of several transplant generations of a GRS/A mouse mammary tumor line was studied. Cyclophosphamide caused more growth inhibition in transplant generation 6 than in other transplant generations. Transplant generation 6 represented the time of transition in the tumor line from hormone responsiveness to hormone independence. These results suggest that mammary tumors are especially sensitive to chemotherapy at the stage when the first hormone-independent cells appear in the tumor.

Sex hormone receptors in mammary tumours of GR mice.

Studies of the growth behaviour and hormone receptor contents of GR-mouse mammary tumours suggest that the hormone responsive tumours are mixed populations of hormone-dependent cells and autonomous cells. The hormone-dependent moiety of these tumours contains high levels of progesterone receptor and oestrogen receptor. The autonomous moiety has a low but probably significant oestrogen receptor level, and is practically devoid of progesterone receptor. Androgen receptor levels in both moieties are low. Endocrine ablation prevents growth of the hormone-dependent moiety of the tumours, but not of the autonomous moiety.