Cytotoxicity of 125I-oestrogen decay in non-oestrogen receptor-expressing human breast cancer cells, MDA-231 and oestrogen receptor-expressing MCF-7 cells.

PURPOSE: To compare the cytotoxicity of 125I-oestrogen (E-17alpha[125I]iodovinyl-11betamethoxyoestradiol or 125IVME2) decay accumulation in human breast adenocarcinoma cells that do not express oestrogen receptor (ER) (MDA-231 cells) with human breast adenocarcinoma cells that do express ER (MCF-7 cells). MATERIALS AND METHODS: MDA-231 cells were labelled with 125IVME2 or [125I]iododeoxyuridine (125IdU), frozen for decay accumulation, thawed and then plated for colony formation. gamma-irradiation survival was also determined. A whole-cell 3H-oestrogen-binding assay and a specific-binding assay were used to detect ER. RESULTS: No MDA-231 cell killing by accumulated 125IVME2 decays (up to 440 dpc) was observed but ER-positive MCF-7 cells were killed by 125IVME2 (D(o)=28 dpc). MDA-231 cells were not significantly more radioresistant to gamma-rays (D(o)=1.7Gy for MDA-231 cells; 1 Gy for MCF-7 cells) or to 125IdU decays (D(o)= 44dpc for MDA-231 cells; 30 dpc for MCF-7 cells). No ER were detected in MDA-231 cells. CONCLUSIONS: ER-negative cells, MDA-231, are not killed by 125IVME2 decay accumulation. It is speculated that without ER (required to translocate the 125IVME2 to its nuclear target), formation of the 125IVME2-ER-DNA oestrogen-response element (ERE) complex and subsequent specific irradiation of the DNA at the ERE cannot occur. These results support the hypothesis that the nuclear genome is a critical target for radiation-induced cell death.

Therapy of estrogen receptor-positive micrometastases in the peritoneal cavity with Auger electron-emitting estrogens--theoretical and practical considerations.

Previous studies have demonstrated that Auger electron-emitting estrogens, when associated with the estrogen receptor (ER), can effect breaks in DNA and ER-dependent radiotoxicity. To evaluate the potential of [123I]-iodoestrogens, ([123I]-IE) to treat ER-positive human cancer cells, we have studied the effect of incubation of [123I]-IE with ER-positive MCF-7 breast cancer cells on cell survival in vitro and found that subnanomolar concentrations of [123I]-IE effectively reduce survival, with a mean lethal dose of about 800 decays per cell. MCF-7 cells incubated 30 min with 2 nM [123I]-IE (13 MBq/ml) showed a 2 log reduction in the ability to form tumors in immunodeficient mice. Evaluation of a mathematical model for [123I]-IE therapy for intraperitoneal micrometases in vivo in the mouse, based on variables related to the (a) specific activity of [123I]-IE; (b) its affinity for ER; (c) the characteristics of the uptake and retention of [123I]-IE by the target cells; (d) the concentration of ER in the tumor cells and (e) the tumor weight suggest that such therapy may be feasible.

Radiotoxicity of Auger electron-emitting estrogens in MCF-7 spheroids: a potential treatment for estrogen receptor-positive tumors.

To approach treatment of micrometastases of steroid receptor-rich cancers using estrogen receptor-directed therapy with Auger electrons, multicellular spheroids of the estrogen receptor-rich human breast cancer cell line, MCF-7, were prepared and exposed to a range of concentrations of an Auger electron-emitting estrogen, E-17alpha-[123I]-iodovinyl-11beta-methoxyestradiol, [123I]IVME2, in vitro. After washing, the treated spheroids were dissociated to single cells and plated for assay of colony survival, whereby we observed a dose-dependent reduction in survival that was inhibited by inclusion of an excess of unlabeled estradiol in the initial incubation with [123I]IVME2. Spheroids of a range of sizes from 40 to 280 microm showed similar sensitivity to the Auger electron-emitting estrogen. The mean lethal dose was approximately 700 decays per cell and corresponded to an initial [123I]IVME2 concentration of less than 0.5 nM. If the control and treated spheroids were not trypsinized but rather were allowed to grow intact, there was not only a significant reduction in the growth of the treated spheroids, but in 18 days nearly half became necrotic, while few control spheroids were necrotic. Considering the low concentrations of Auger electron-emitting estrogen required for a significant reduction in survival, we believe this approach has merit to pursue in vivo, especially in cases where it may be possible to target the steroid receptor-rich micrometastases directly, such as ovarian cancer.

Uptake and interconversion of the Z and E isomers of 17 alpha-iodovinyl-11 beta-methoxyestradiol in the immature female rat.

This study compares the specific uptake and retention of the Z and E isomers of 17 alpha-iodovinyl-11 beta-methoxyestradiol (IVME2) in estrogen target tissues in immature female rats following intraperitoneal injection. Estrogen receptor binding studies in vitro showed that the Z-IVME2 had greater affinity than the E-IVME2, but our initial in vivo data, comparing 125I-labeled E- or Z-IVME2 in separate studies showed no difference between the two isomers in either uptake or retention. These results were confirmed when the two isomers, labeled either with 123I or 125I, were injected simultaneously, allowing direct comparison of uptake and retention patterns in the same animal. Analysis of the nature of the radioiodinated estrogens in estrogen target tissues showed that at all time points, the estrogen target tissues contained mainly the E isomer, even at short times after injection of Z isomer. Although the Z-IVME2 was reasonably stable in the injectant, analysis of a peritoneal lavage soon after intraperitoneal injection of Z-[125I]-IVME2 showed that mainly the E isomer was present, suggesting that the conversion occurred prior to uptake by the tissues. In vitro studies with intraperitoneal fluid and serum showed that denaturing by heating at 65 degrees C substantially reduced their ability to affect the conversion of Z to E isomer, suggesting a possible enzymatic conversion.

DNA and chromosome breaks induced by iodine-123-labeled estrogen in Chinese hamster ovary cells.

The effects of the Auger electron-emitting isotope 123I, covalently bound to estrogen, on DNA single- and double-strand breakage and on chromosome breakage was determined in estrogen receptor-positive Chinese hamster ovary (CHO-ER) cells. Exposure to the 123I-labeled estrogen induced both single- and double-strand breaks with a ratio of single- to double-strand breaks of 2.8. The corresponding ratio with 60Co gamma rays was 15.6. The dose response was biphasic, suggesting either that receptor sites are saturated at high doses, or that there is a nonrandom distribution of breaks induced by the 123I-labeled estrogen. The 123I-labeled estrogen treatment induced chromosome aberrations with an efficiency of about 1 aberration for each 1000 disintegrations per cell. This corresponds to the mean lethal dose of 123I-labeled estrogen for these cells, suggesting that the lethal event induced by the Auger electron emitter bound to estrogen is a chromosome aberration. Most of the chromosome-type aberrations were dicentrics and rings, suggesting that 123I-labeled estrogen-induced chromosome breaks are rejoined. The F ratio, the ratio of dicentrics to centric rings, was 5.8 +/- 1.7, which is similar to that seen with high-LET radiations. Our results suggest that 123I bound to estrogen is an efficient clastogenic agent, the cytotoxic damage produced by 123I bound to estrogen is very like damage induced by high-LET radiation, and the 123I in the estrogen receptor-DNA complex is probably in proximity to the sugar-phosphate backbone of the DNA.

DNA damage induction by 125I-estrogen.

DNA damage induced by the radioactive decay of 125I-estrogen (125I-VME2) in an estrogen receptor expressing CHO cell line, CHO-ER, was measured. 125I-VME2 targeted 125I atoms proximal to DNA estrogen response elements (EREs). 125I decays were accumulated at -135 degrees C, and thereafter assayed by alkaline and neutral filter elution techniques to measure DNA single strand break (ssb) and double strand break (dsb) induction respectively. Increasing DNA damage (both ssbs and dsbs) was detected after exposure of cells to increasing concentrations of 125I-VME2. DNA ssb and dsb dose-response curves for 125I-VME2 were multiphasic. The rates of DNA damage induction by the decay of 125I-VME2 was determined by comparing slopes of all data or by comparing initial slopes. DNA ssb induction per 125I-VME2 decay was approximately 2 times greater compared with DNA dsb induction. 125I-VME2 decay induced approximately 4-8 times more DNA dsbs than 125IUdR decay.

Cellular and subcellular studies of the radiation effects of Auger electron-emitting estrogens.

We studied the effect of 123I-labeled estrogen (123I-E) in estrogen receptor (ER)-rich cells in culture and in cell free model systems in vitro to elucidate the nature of the radiotoxicity for ER + cells of estrogens containing nuclides which emit Auger electrons. In cells the 123I-E caused a dose-dependent, unlabeled estrogen-inhibitable induction of chromosome aberrations. A dose of about 1000 decays per cell, which is approximately the mean lethal dose for these cells, resulted in an average of 1 chromosome break per cell. This supports the hypothesis that the lethal lesion induced by 123I-E is a chromosome break. Incubation of 123I-E/ER complex, but not 123I-E alone, with 27-mer duplex estrogen response element (ERE) DNA produced a dose-dependent cleavage of the ERE. However, we were unable to detect any fragmentation of either the 66 kDa full length ER in cell extracts or a purified 31 kDa hormone binding domain when incubated with excess 123I-E. Thus it appears that 123I-E effects its radiotoxicity by binding to ER, associating with ERE DNA and, by directing high LET radiation to DNA, inducing lethal chromosome breaks.

Comparison of the distribution of radioiodinated di- and tri-hydroxyphenylethylene estrogens in the immature female rat.

The uptake, retention and tissue to blood ratios of two non-steroidal, 125I-labeled iodoestrogens, an iodotrihydroxyphenylethylene, 2-iodo-1,2-bis(4-hydroxyphenyl)-2-(3-hydroxyphenyl)ethylene and an iododihydroxyphenylethylene, 2-iodo-1,1-bis(4-hydroxyphenyl)-2-phenylethylene, were compared after intraperitoneal injection in immature female rats. The iodotrihydroxyphenylethylene showed an unexpectedly prolonged specific retention in estrogen target tissue, lasting up to 72 h. It was rapidly cleared from blood and non-target tissues so that uterus or ovary to blood ratios of greater than 100 were seen at 2 and 3 days. This iodotrihydroxyphenylethylene may have clinical potential for estrogen receptor-containing cancers.

Immunohistochemical localization of the oestrogen-responsive 110 kDa and 74 kDa polypeptides and complement component C3 in the rat genital tract after oestrogen treatment and during the oestrous cycle.

In previous studies we showed that two oestrogen-responsive rat uterine secretory polypeptides of molecular mass 110 and 74 kDa share sequence similarities with the alpha and beta chains of rat complement component C3. Using polyclonal antibodies specific to the 110 kDa polypeptide, the 74 kDa polypeptide and rat serum C3, we studied the localization of these proteins in the rat genital tract using an indirect immunoperoxidase technique. The results showed that in oestradiol-treated rats immunostaining for the three antigens was heterogeneously distributed in the epithelia of the endometrium, cervix and vagina and that the intensity of staining was greater after oestrogen treatment than in controls. In oestradiol-treated immature rats, the immunoreactivity for the 110 kDa and 74 kDa polypeptides was greater in the endometrial and vaginal epithelia than in the cervical epithelium, whereas immunoreactivity for C3 was greatest in the vaginal epithelium. In cyclic rats, staining by all three antibodies was seen only during pro-oestrus and oestrus, during which stages the immunostaining for the 110 kDa and 74 kDa polypeptides was detected only in the endometrial epithelium, whereas immunostaining for C3 was also found in the epithelia of the cervix and vagina. In general, consecutive sections of these tissues revealed a close correlation between the immunostaining for the 110 kDa and 74 kDa polypeptides and C3. However, there were some sections that showed clear differences in staining, suggesting that more than one C3-related protein species was detected in the female rat reproductive tract.

Comparison of the distribution of radioiodinated-E-17 alpha-iodovinyl-11 beta-methoxyestradiol and 2-iodo-1,1-bis(4-hydroxyphenyl)-phenylethylene estrogens in the immature female rat.

Current therapies for estrogen receptor-positive (ER+) cancers rely heavily on growth prevention by cytostatic agents rather than destruction of the cancer cells. In these studies, we compared the tissue distribution of two radioiodinated estrogens, one a triphenylethylene estrogen, IBHPE, the other steroidal, IVME2. The radioiodoestrogens were prepared using the halodestannylation reaction from the respective tributyltin precursors. The specificity of binding of these radioiodinated estrogens to the estrogen receptor (ER) was established by sucrose-density gradient analyses and their specific activities by comparison with the ER binding of 3H-estradiol of known specific activity. The time-dependent tissue distribution of the two radioiodoestrogens in immature female rats was studied to compare the relative uptake and specific retention of the two estrogens in ER target organs and assess their possible use for imaging ER positive tissues or for Auger electron-mediated ER-directed therapy. While the uterus showed only slightly poorer retention of the nonsteroidal estrogen (IBHPE) than the steroidal estrogen (IVME2), those target tissues that required blood-supplied ligand (e.g., vagina, pituitary) showed substantially higher uptake of the steroidal estrogen. IBHPE showed significantly higher blood levels at all time points. While the tissue-to-muscle ratios for IBHPE in the uterus and ovary were higher initially, the IVME2 showed higher tissue-to-muscle ratios, suggesting that the steroidal estrogen may be the more promising ligand for imaging purposes. However, IBHPE showed excellent uptake by peritoneal target tissues, with much lower concentrations in more distant target tissues (e.g., pituitary) so it might have distinct potential for therapy of intraperitoneal ER-containing cancers.

Estrogen receptor-directed radiotoxicity with Auger electrons: specificity and mean lethal dose.

To assess the feasibility of using estrogen receptor-directed therapy with Auger electron-emitting ligands for therapy of estrogen receptor (ER)-containing cancers, we synthesized and evaluated the radiotoxicity of several 123I-labeled estrogens to specifically kill ER+ cells in culture. Auger electrons have been previously shown to be of short range, generally less than the dimensions of a cell, so that to use them therapeutically a mechanism is needed to deliver the Auger electron-emitting nuclide to the vicinity of the DNA. Since it is now well established that the estrogen receptor, when bound to estrogen, forms a high affinity association with distinct estrogen response elements in the DNA, we wished to test the hypothesis that a short exposure of cells to a 123I-labeled estrogen would be specifically radiotoxic to ER+ cells, and that the decays per cell needed for cell killing would be compatible with reasonable levels of receptor occupancy. Using the halodestannylation reaction with tributyl tin precursors of several estrogens and commercially available iodine-123, we prepared the iodoestrogens, E-17 alpha(-)[123I]-iodo-11 beta-methoxyestradiol and 2(-)[123I]iodo-1,1-bis(4-hydroxyphenyl)--2-phenylethylene, at high specific activities, in several cases at essentially the specific activity of 123I itself, 240,000 Ci/mmol. When various concentrations of either of the 123I-labeled estrogens were incubated for 1 h with a subline of ER+ Chinese hamster ovary cells and the washed cells plated for survival assays, a dose-dependent, unlabeled estradiol-inhibitable reduction in survival was observed. In contrast, Chinese hamster ovary cells not expressing estrogen receptor showed little sensitivity to the radiotoxicity of the 123I-labeled estrogens. Calculations based on the assayed residence time of the iodoestrogens in the cells indicate that several hundred decays per cell are sufficient to kill cells.

Synthesis, purification and stability of no carrier added radioiodinated 1,1-bis(4-hydroxyphenyl)-2-iodo-2-phenylethylene (IBHPE), a prototype triphenylethylene estrogen-receptor binding radiopharmaceutical.

A triphenylethylene compound [1,1-bis(4-hydroxyphenyl)-2-iodo-2-phenylethylene; IBHPE] has been labeled by halodestannylation with 123I at a specific radioactivity of 13,200 Ci/mmol (by in vitro receptor assay) after HPLC purification. The corresponding 80mBr-labeled compound (BrBHPE), which has a 3-fold higher affinity for the estrogen receptor, was previously prepared and examined as a potential therapeutic radiopharmaceutical exploiting Auger electron toxicity. Stability of IBHPE was a concern because free iodide was generated when HPLC solvents were removed with a stream of nitrogen in a glass vial; however, decomposition was minimal when polypropylene vials were used, and ethanol solutions of [123I]IBHPE were stable for several days at 0-4 degrees C. Tissue distribution studies of IBHPE after intraperitoneal injection to mature female rats showed highest estradiol-inhibitable uptake in the peritoneal estrogen-receptor rich tissues (uterus, ovaries and vagina) at 30 min. Specific uptake (percent dose per gram) in the pituitary, and peritoneal target tissue-to-blood ratios were greater at 2 h than 30 min. In immature female rats, uterus-to-blood ratios of greater than 50, progressively lowered by increasing diethylstilbestrol levels, were obtained. These data demonstrate good binding of IBHPE to the estrogen receptor in vivo, in spite of extensive non specific binding in in vitro estrogen receptor assays. Most of the label in the uterus at 1 h after injection was still unchanged IBHPE. Our results suggest that IBHPE or related 123I-labeled iodovinyl triphenylethylenes could have diagnostic or therapeutic radiopharmaceutical utility.

Comparison of the distribution of bromine-77-bromovinyl steroidal and triphenylethylene estrogens in the immature rat.

The specific uptake and distribution of steroidal and non-steroidal [77Br]bromovinylestrogens were studied in immature female rats to assess the potential of these radioligands for imaging or therapy of estrogen receptor (ER) positive cancers. E-17 alpha [77Br]bromovinylestradiol, its 3 methyl ether and 11 beta-methoxy derivative, as well as the triphenylethylene estrogen, 1,1-bis[4-hydroxy-phenyl]-2-[77Br]bromo-2- phenylethylene all showed diethylstilbestrol inhibitable, specific uptake of radiobromine between 2 and 16 hr after i.p. administration. The highest concentrations in the estrogen target tissues and the highest target tissue-to-blood ratios were found with E-17 alpha-[Br]bromovinyl-11 beta-methoxyestradiol, but it also had rather high nonspecific uptake in all tissues. The triphenylethylene estrogen showed comparable specific uptake in estrogen target tissues to 17 alpha [77Br]bromovinylestradiol at 2 hr but better apparent retention, indicated by higher specific target tissue levels at the later time points. Thus, [77Br]bromovinyl-11 beta- methoxyestradiol and 1,1-bis[4-hydroxyphenyl]-2-[77Br]bromo-2-phenylethylene appear most favorable for these applications.

Comparison of the quantity of estrogen receptors in human endometrium and myometrium by steroid-binding assay and enzyme immunoassay based on monoclonal antibodies to human estrophilin.

The recently developed enzyme immunoassay for estrogen receptors is more simple to perform with quality assurance than conventional steroid-binding assays with radioactive labeled estrogen. However, it is not known how well the results of the two assays agree for normal human uterine samples. We compared enzyme immunoassay (Abbott estrogen receptor enzyme immunoassay) and steroid-binding assay of normal human endometrium and myometrium. Low-salt, "cytosol" estrogen receptor determinations were performed by dextran-coated charcoal assay, and high-salt, "nuclear" estrogen receptors were measured by controlled pore glass bead assay. Results showed excellent correlation (p less than 0.0001) for cytosol estrogen receptor of endometrium (r = 0.95) and myometrium (r = 0.79) and also for a smaller number of nuclear estrogen receptors of myometrium (p less than 0.01, r = 0.69). Good agreement between steroid-binding assay and enzyme immunoassay was seen for estrogen receptors of both proliferative and secretory phase samples. Thus the data indicate that the simpler estrogen receptor enzyme immunoassay is useful to measure estrogen receptor in the normal uterus. Furthermore, with this sandwich assay, there is no evidence for the existence of significant quantities of receptor fragments that do not bind estrogen.

The estrogen-responsive 110K and 74K rat uterine secretory proteins are structurally related to complement component C3.

Estrogens stimulate the synthesis of specific secretory proteins in the rat uterus. Here we show that two of these, polypeptides of relative molecular weight 110,000 (110K) and 74,000 (74K), are structurally related to C3, the third component of complement, a glycoprotein that plays a central role in regulating complement-mediated inflammatory and immune responses. The similarities were based on the observations that (1) NH2-terminal amino acid sequence of the 74K polypeptide showed sequence homology with the beta chain of mouse C3, (2) comparison of the electrophoretic mobilities of the 110K and 74K polypeptides in the presence and absence of reducing agents revealed that they were disulfide-linked subunits of a protein of Mr approximately 180,000, (3) the native protein was immunoreactive with antibodies specific for rat C3, and (4) both polypeptides were immunoprecipitated with antibodies to rat C3.

Bromine-80m radiotoxicity and the potential for estrogen receptor-directed therapy with auger electrons.

While theoretically feasible, estrogen receptor (ER)-directed radiotherapy of hormone-dependent cancers has not been realized because no ER-seeking ligand with an appropriate radiotoxic potential has been identified. Since an appropriate nuclide is a key component we studied the 4.4-h half-life, Auger electron-emitting nuclide bromine-80m. When incorporated into DNA this nuclide was radiotoxic to cells in culture and caused substantial chromosomal damage, while similar concentrations of bromine-80m as bromide or bromoantipyrine were without effect. The mean lethal dose for bromine-80m was 45 atoms per nucleus which is consistent with use in receptor-positive cancers with limited numbers of ER.