Compositions and methods for demonstrating secretory immune system regulation of steroid hormone responsive cancer cell growth
Serum-containing and serum-free immunoglobulin inhibitors of steroid hormone responsive cancer cell growth are disclosed, along with their methods of production. Also disclosed are defined cell culture media, assay protocols, and model systems using the inhibitors for demonstrating steroid hormone growth effects of natural and synthetic substances, and other cell culture applications. The disclosed compositions and methods employing the immunoglobulin inhibitors are also useful as reagents in research, and for the diagnosis, treatment and prevention of mucus epithelial cancers.
1. An in vitro assay method for detecting cancer cell growth stimulation by a substance of interest, the method comprising:
maintaining a predetermined population of steroid hormone-responsive mucosal epithelial cancer cells in a steroid hormone-free nutrient medium comprising a basal nutrient fluid devoid of unbound Fe (III) and comprising calcium ions and an amount of at least one immunoglobulin chosen from the group consisting of non-monomeric plasma IgA and polymeric IgM sufficient to inhibit cell growth in the absence of an inhibition-reversing amount of a steroid hormone, said cells also being steroid hormone dependent for proliferation in vivo when implanted into a suitable host;
adding said substance of interest to said cells and nutrient medium to yield a test mixture;
incubating said test mixture for a predetermined period of time under cell growth promoting conditions; and
determining the cell population in said test mixture after said predetermined period of time, a measurable increase in said cell population indicating a cancer cell growth stimulating effect by said substance of interest.
2. The assay method of claim 1 comprising maintaining serum-free assay conditions.
3. The assay method of claim 1 wherein the nutrient medium further includes steroid-hormone depleted serum.
4. The assay method of claim 1 wherein the nutrient medium further includes serum that has not been subjected to heat inactivation.
5. The assay method of claim 1 wherein said immunoglobulin is polymeric IgM.
6. The assay method of claim 1 wherein said substance of interest is suspected of containing proteolytic activity, in which said immunoglobulin resists protease degradation.
7. The assay method of claim 1 wherein said immunoglobulin is non-monomeric plasma IgA.
8. The assay method of claim 1 further comprising:
maintaining a second predetermined population of said steroid hormone-responsive mucosal epithelial cancer cells in a steroid hormone-free nutrient medium, said cells also being steroid hormone responsive for proliferation in vivo when implanted into a suitable host;
adding said substance of interest to said cells and nutrient medium, to yield a control mixture;
incubating said control mixture for a predetermined period of time under cell growth promoting conditions;
determining the cell population in said control mixture after said predetermined period of time, a measurable increase in said cell population indicating a control level cell growth stimulating effect by said substance of interest.
9. The method of claim 1 comprising:
maintaining a predetermined population of estrogen responsive mucosal epithelial cancer cells in a steroid hormone-free nutrient medium comprising an amount of at least one immunoglobulin chosen from the group consisting of non-monomeric plasma IgA and polymeric IgM sufficient to inhibit cancer growth in the absence of an inhibition-reversing amount of estrogen, said cell also being estrogen responsive for proliferation in vivo when implanted into a suitable host;
adding a defined amount of said substance of interest to said cells and medium, to yield a test culture;
incubating said test culture for a predetermined period of time under cell growth promoting conditions; and
determining the cell population in said test culture after said predetermined period of time, a measurable increase in said cell population indicating cell growth stimulating effect by said substance of interest, whereby an estrogenic substance is detected.
10. The method of claim 9 further comprising testing said substance of interest for cytotoxic effects on said cells.
11. The method of claim 1 wherein said nutrient medium comprises a Fe (III) chelating agent.
12. The method of claim 1 wherein said nutrient medium comprises a cell attachment promoting protein.
13. The method of claim 1 wherein said nutrient medium contains about 1-50 mM calcium ion.
14. The method of claim 1 wherein said basal nutrient fluid comprises D-MEM/F-12.
15. The method of claim 1 wherein said nutrient medium comprises 100 ng/mL to 10 μg/mL insulin, 0.3-10 nM triiodothyronine, 2- 50μg/mL diferric transferrin, 5-100 μM ethanolamine, 0.2-5.0 mg/mL bovine serum albumin (BSA), 5-20 ng/mL selenium, 2-10 μM deferoxamine, and, optionally, at least one component chosen from the group consisting of 1-50ng/mL EGF, 0.2-20ng/mL aFGF, 5-50μM phosphoethanolamine, 50-500 μg/mL linoleic acid-BSA, 1-50 μg/mL reduced glutathione, 0.5-2.0 mM glutamine, 1-10 μg/mL heparin, and 20-50 μg human fibronectin.
16. The assay method of claim 1 wherein the steroid hormone-responsive mucosal epithelial cancer cells are selected from the group consisting of GH4C1, GH1 and GH3 rat pituitary cells, ZR-75-1 human breast cancer cells, MCF-7A human breast cancer cells, T47D human breast cancer cells, LNCaP human prostate cancer cells, and HT-29human colon cancer cells.
17. The assay method of claim 1 wherein the steroid hormone is selected from the group consisting of Estrogens, Androgens, Progesterone, and Glucocorticoids.
18. The assay method of claim 17 wherein the steroid hormone-responsive mucosal epithelial cancer cells are selected from the group consisting of GH4C1, GH1 and GH3 rat pituitary cells, ZR-75-1 human breast cancer cells, MCF-7A human breast cancer cells, T47D human breast cancer cells, LNCaP human prostate cancer cells, and HT-29human colon cancer cells.
19. The assay method of claim 3 wherein the steroid hormone-responsive mucosal epithelial cancer cells are selected from the group consisting of GH4C1, GH1 and GH3 rat pituitary cells, ZR-75-1 human breast cancer cells, MCF-7A human breast cancer cells, T47D human breast cancer cells, LNCaP human prostate cancer cells, and HT-29human colon cancer cells.
20. The assay method of claim 3 wherein the steroid hormone is selected from the group consisting of Estrogens, Androgens, Progesterone, and Glucocorticoids.
21. The assay method of claim 20 wherein the steroid hormone-responsive mucosal epithelial cancer cells are selected from the group consisting of GH4C1, GH1 and GH3 rat pituitary cells, ZR-75-1 human breast cancer cells, MCF-7A human breast cancer cells, T47D human breast cancer cells, LNCaP human prostate cancer cells, and HT-29human colon cancer cells.
22. An in vitro assay method for detecting steroid hormone cancer cell growth stimulation by a substance of interest, the method comprising:
maintaining a predetermined population of steroid hormone-responsive mucosal epithelial cancer cells in a steroid hormone-free nutrient medium comprising a basal nutrient fluid devoid of unbound Fe (III) and comprising calcium ions and an amount of at least one immunoglobulin chosen from the group consisting of non-monomeric plasma IgA and polymeric IgM sufficient to inhibit cell growth in the absence of an inhibition-reversing amount of a steroid hormone, said cells also being steroid hormone dependent for proliferation in vivo when implanted into a suitable host;
adding said substance of interest to said cells and medium to yield a test mixture;
incubating said test mixture for a predetermined period of time under cell growth promoting conditions; and
determining the cell population in said test mixture after said predetermined period of time, wherein a measurable increase in said cell population indicates a steroid hormone dependent cancer cell growth stimulating effect by said substance of interest.
23. The method of claim 22 wherein the non-monomeric plasma IgA is dimeric/polymeric IgA.
24. An in vitro assay method for detecting estrogenic cancer cell growth stimulation by a substance of interest, the method comprising:
maintaining a predetermined population of estrogen-responsive mucosal epithelial cancer cells in a steroid hormone-free nutrient medium comprising a basal nutrient fluid devoid of unbound Fe (III) and comprising calcium ions and an amount of at least one immunoglobulin chosen from the group consisting of non-monomeric plasma IgA and polymeric IgM sufficient to inhibit cell growth in the absence of an inhibition-reversing amount of an estrogen, said cells also being estrogen dependent for proliferation in vivo when implanted into a suitable host;
adding said substance of interest to said cells and medium to yield a test mixture;
incubating said test mixture for a predetermined period of time under cell growth promoting conditions; and
determining the cell population in said test mixture after said predetermined period of time, wherein a measurable increase in said cell population indicates an estrogenic dependent cancer cell growth stimulating effect by said substance of interest.
25. The method of claim 24 wherein the non-monomeric plasma IgA is dimeric/polymeric IgA.
26. The method of claim 7 wherein the non-monomeric plasma IgA is dimeric/polymeric IgA.