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Estrogenic effects of nonylphenol on PS2, MUC1 and ER expressions in human breast cancer cells MCF-7
Abstracts
ESTROGENIC EFFECTS OF NONYLPHENOL ON PS2, MUC1 AND ER EXPRESSIONS IN HUMAN BREAST CANCER CELLS MCF-7 R.L.R~,MA. Marquardi and JJ. Etch, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, W153226. '1~ r"
D~
Over the last fifty years, large amounts of estrogenic man-made chemicals have been inadvertently released into the environment. Many chemicals have recently been discovered to have estrogenic activity, including the surfaetant intermediate nonylphanol (NP). It has been well documented that estrogen is a facilitator of human breast cancer development and environmental estrogens such as NP have recently been under intense investigation. The reason we used the expression of PS2, MUCI and estrogen receptor (ER) genes as markers of estrogenic effects are based on 1) PS2 is an estrogen induced gene product first identified in die MCF-7 human breast cancer cell line after estrogen treatmenL but not found in normal breast tissue nor in any other cultured human cell lines. 2) MUC1 is a member of a family of high molecular weight, highly glycosylatad rnucin glycoproteins found in the membranes of human epithelial cells. Dysfunction of mucin, such as MUCI overexpressinn, is believed to play important roles in mammary gland tumorigenesis. MUCI expression is also induced by estrogen. 3) It is widely accepted that estrogen regulates target gene expression through the specific binding of estrogen receptor. The ER/estrogen complex is able to modulate transcriptional activity by interacting with the estrogen responsive element in target promotor genes. ER gene expression is also affected by estrogen. Results: The time course of mRNA expressions were detected after NP (10 pM) and estradiol (E2) (0.1 ~tM) treatments using RT-PCR technique. The products were run on a 1.6% agarose gel and quantitatad with the Finorlmager. 1) NP induced pS2 mRNA after a two hour exposure, similar to E2 induction. At 24 hours, the total RNA was increased 2.4 and 4.2-fold, respectively, with NP and E2 treatmem, indicating posalble induction of MCF-7 cell growth. 2) The highest level of MUCl mRNA seen after NP tre~mem was at 2 hours ( 10-fold vs. control), which was reduced to only 42% of control at 48 hr. E2 treatment resulted in a gradual increase in MUCI expression; double the control at 2 hr, 54old increase at 24 hr and increased by a factor of 10 over control at 48 hr. This indicates that NP may stimulate MUC1 expression by a different mechanism than E2. 3) NP affected ER expression in the same manner as MUC1 ; the highest level of expression occurred at 2 hi', but expression was gradually reduced to control level by 48 hr, In contrast, E2 stimulated ER expression in a similar manner as pS2; the highest level was at 2 hr and expression remained eleva~l through the 48 hr point. Dlscumlon: NP is an estrogenic compound that alters PS2, MUC1 and ER gene expression, possibly by a different mechanism than E2.~ According to recent dfiterature,6 however, the mechanism bycewhich E2 and NP regulate gene expression is tistill anclear,6 as is die role of ER in the process. Sincexeinduction begins atter a 2 hour exposure, it has been suggested that a post/~Itranseriptional regulation pl~cessommy ha involved. The involvement of estrogen in RNA processing is cttrt ently under invesdgatinn in ourmlab. The link between aberrant MUCI, PS2 and ER expression and die development of breast cancer also needs to be elucidated through further investigation. ]l~t" uu ~ , NM
W H O L E A R M T R A N S L O C A T I O N S IN S O L I D T U M O R S A N D HEMATOLOGIC MALIGNANCY - FORMATION AND CLINICAL SIGNIFICANCE, Lytle C, Lamb A, Velasco J, Roherty S. Genzyme Genetics, Santa Fe,
Whole ann translocatiuns (WAT) involve breakage and reunion of the pericentromeric regions of nonhomniognus chromosomes. Robertsoniau translocations involving acrocentric chromosomes are common constitutional rearrangements, but are rare as acquired changes in neoplastic diseases, where nonacrocentric translocations occur more frequently. WATs may be balanced or, i f unbalanced, produce partial monosomies or trisomies for the affected chromosomes. FISH analyses with a-satellite probes have been used in some cases to determine breakpoints and indicate mechanism o f formation of WATs. We have reviewed over 20,000 cytogenetic analyses o f patients with hematologic malignancy or solid tumors referred to our laboratory over a 14-year period to determine the incidence of WATs and to assess their clinical significance. 175 cases were identified, suggesting an incidence o f about 3% in cytogenetically abnormal cases. FISH analyses with a-satellite probes were performed on 28 cases to determine the origin of the centromeric regions of the derived chromosomes. WATs were observed in lymphoid, myeloid and solid tumors, and occurred more often in males than in females. Balanced WATs were ohserved as the sole change in 10 cases, and as part of a more complex karyotype in an additional 2 6 cases. Unhalauced WATs were seen in 142 cases, with the most common resulting in imbalance for chromosome 1 (38 cases), chromosome 7 (37 cases) and chromosome 17 (34 cases). F I S H analyses indicated that 20 cases had centromeric material from both chromosomes involved in the WAT, while 8 cases had centromeric material from only one chromosome detectable by FISH. The existence of balanced WATs as sole changes, the presence o f WATs in patients with biclonal disorders, and their frequent observation in complex karyotypes in patients with secondary disease suggest that, in addition to being pathognomic in some patients, they may also provide a general measure o f genomic instability associated with neoplastic development aud/or exposure to clastogenic/earcinogenic agents.
165
B7
CYTOGENETIC ANALYSIS OF A GLIOBLASTOMA IN A PATIENT WITH TURCOT SYNDROME. Roherty. SE, Richldnd, KE, Chilcote R. Genzyme Genetics, Santa Fe, N M (SER, KER); UCi, Irvine, C A (RC) Turcot syndrome is characterized by the concurrence o f colorectal tumors and primary brain tumors, with other tumor types also occurring. Familial cases have been associated with germliue mutations of the APC, hMI,HI or hPMS2 genes. The brain tumors found in these families are primarily medunoblastomas (APC gene mutations) or glioblastomas (HNPCC geue mutations). We describe the first cytogenetic study o f a glioblastoma associated with Tureot syndrome. The tumor was surgically reseeted from a 19-year-old female patient, who had a history o f intestinal polyps and, at age 14, had been diagnosed with fibrnsarcoma and treated with radiation and VP- 16. Cytogenetically the giioblastoma was characterized by clonal heterogeneity, with four pseudodiploid clones present. There was a preponderance of balanced structural rearrangements, with two o f the four clones demonstrating only balanced rearrangements. The other two clones had imbalances affecting chromosome 3, as well as a number of balanced translocations. Three of the four clones had jumping translocations involving 19q13. This tumor was very aggressive and recurred within six weeks, and the patient died soon after. The pattern of abnormalities observed in this tumor is unusual but is more similar to that reported for recurrent gliomas rather than for primary gliomas, w h i c h are more often aueuploid, pniyplnid and have unbalanced structural rearrangements. Whether this pattern is typical of glioblastomas associated with Turcot syndrome is unknown, but potential association between possible radiosensitivity, replication error repair mutations and cytogenetic abnormalities warrants further investigation.
B8
UNUSUAL KARYOTYPES IN TWO SYNOVIAL SARCOMAS. Roland. B. Departments o f Pathology and Medical Genetics, University o f Calgary, Calgary, Alberta, Canada. The characteristic chromosome abnormality in synovial sarcoma is t(X;18)(pl 1.2;ql 1.2), which is observed in over 80% o f abnormal karyotypod synovial sarcomas. Secondary chromosome abnormalities are frequent, with 75% of reported cases having structural aud/or numerical abnormalities in addition to t(X; 18). In this report, two synovial sarcomas with unusual karyotypes are described. The first case is a 22 cm primary thigh mass with spindle cell morphology from a 24 year old female. The tumour has a near-triploid karyotype o f 66-75,X,-X, t(X;18)(pll.2;qll.2),+2,+4,+5,-7,+9,+12,+14,-15,+22,+3mar[cp3]/46,XX[21. The second case is a primary lung mass with monomorphic spindle cell morphology, from a 46 year old female. The tumour kas a karyotype of 45,X,t(X;3; 18)(p l l . 2 ; q 2 9 ; q l 1.2),- 18113]. Clinical follow-up in both cases is less than 6 months. These two tumours are of interest because they have the typical U'anslocation for synovial sarcoma, hut also have unique chromosome abnormalities. T h e majority of cases of synovial sarcoma previously reported have been near diploid, with oniy three previous turnouts having chromosome counts above the hyperdiploid range. The tumunr presented here is the second in the near-triploid range. Several 3-way trauslocations involving chromosomes X, 18 and another autosome have been reported previously. The loss o f the normal chromosome 18 in the second case reported here, leaving only the tranalocated 18, is however, unique. Further tumour karyotypes with clinical correlation are required to determine the significance o f the near-triploid karyotype and the loss o f the normal chromosome 18.
ESTROGENIC EFFECTS OF NONYLPHENOL ON PS2, MUC1 AND ER EXPRESSIONS IN HUMAN BREAST CANCER CELLS MCF-7 R.L.R~,MA. Marquardi and JJ. Etch, Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, W153226. '1~ r"
D~
Over the last fifty years, large amounts of estrogenic man-made chemicals have been inadvertently released into the environment. Many chemicals have recently been discovered to have estrogenic activity, including the surfaetant intermediate nonylphanol (NP). It has been well documented that estrogen is a facilitator of human breast cancer development and environmental estrogens such as NP have recently been under intense investigation. The reason we used the expression of PS2, MUCI and estrogen receptor (ER) genes as markers of estrogenic effects are based on 1) PS2 is an estrogen induced gene product first identified in die MCF-7 human breast cancer cell line after estrogen treatmenL but not found in normal breast tissue nor in any other cultured human cell lines. 2) MUC1 is a member of a family of high molecular weight, highly glycosylatad rnucin glycoproteins found in the membranes of human epithelial cells. Dysfunction of mucin, such as MUCI overexpressinn, is believed to play important roles in mammary gland tumorigenesis. MUCI expression is also induced by estrogen. 3) It is widely accepted that estrogen regulates target gene expression through the specific binding of estrogen receptor. The ER/estrogen complex is able to modulate transcriptional activity by interacting with the estrogen responsive element in target promotor genes. ER gene expression is also affected by estrogen. Results: The time course of mRNA expressions were detected after NP (10 pM) and estradiol (E2) (0.1 ~tM) treatments using RT-PCR technique. The products were run on a 1.6% agarose gel and quantitatad with the Finorlmager. 1) NP induced pS2 mRNA after a two hour exposure, similar to E2 induction. At 24 hours, the total RNA was increased 2.4 and 4.2-fold, respectively, with NP and E2 treatmem, indicating posalble induction of MCF-7 cell growth. 2) The highest level of MUCl mRNA seen after NP tre~mem was at 2 hours ( 10-fold vs. control), which was reduced to only 42% of control at 48 hr. E2 treatment resulted in a gradual increase in MUCI expression; double the control at 2 hr, 54old increase at 24 hr and increased by a factor of 10 over control at 48 hr. This indicates that NP may stimulate MUC1 expression by a different mechanism than E2. 3) NP affected ER expression in the same manner as MUC1 ; the highest level of expression occurred at 2 hi', but expression was gradually reduced to control level by 48 hr, In contrast, E2 stimulated ER expression in a similar manner as pS2; the highest level was at 2 hr and expression remained eleva~l through the 48 hr point. Dlscumlon: NP is an estrogenic compound that alters PS2, MUC1 and ER gene expression, possibly by a different mechanism than E2.~ According to recent dfiterature,6 however, the mechanism bycewhich E2 and NP regulate gene expression is tistill anclear,6 as is die role of ER in the process. Sincexeinduction begins atter a 2 hour exposure, it has been suggested that a post/~Itranseriptional regulation pl~cessommy ha involved. The involvement of estrogen in RNA processing is cttrt ently under invesdgatinn in ourmlab. The link between aberrant MUCI, PS2 and ER expression and die development of breast cancer also needs to be elucidated through further investigation. ]l~t" uu ~ , NM
W H O L E A R M T R A N S L O C A T I O N S IN S O L I D T U M O R S A N D HEMATOLOGIC MALIGNANCY - FORMATION AND CLINICAL SIGNIFICANCE, Lytle C, Lamb A, Velasco J, Roherty S. Genzyme Genetics, Santa Fe,
Whole ann translocatiuns (WAT) involve breakage and reunion of the pericentromeric regions of nonhomniognus chromosomes. Robertsoniau translocations involving acrocentric chromosomes are common constitutional rearrangements, but are rare as acquired changes in neoplastic diseases, where nonacrocentric translocations occur more frequently. WATs may be balanced or, i f unbalanced, produce partial monosomies or trisomies for the affected chromosomes. FISH analyses with a-satellite probes have been used in some cases to determine breakpoints and indicate mechanism o f formation of WATs. We have reviewed over 20,000 cytogenetic analyses o f patients with hematologic malignancy or solid tumors referred to our laboratory over a 14-year period to determine the incidence of WATs and to assess their clinical significance. 175 cases were identified, suggesting an incidence o f about 3% in cytogenetically abnormal cases. FISH analyses with a-satellite probes were performed on 28 cases to determine the origin of the centromeric regions of the derived chromosomes. WATs were observed in lymphoid, myeloid and solid tumors, and occurred more often in males than in females. Balanced WATs were ohserved as the sole change in 10 cases, and as part of a more complex karyotype in an additional 2 6 cases. Unhalauced WATs were seen in 142 cases, with the most common resulting in imbalance for chromosome 1 (38 cases), chromosome 7 (37 cases) and chromosome 17 (34 cases). F I S H analyses indicated that 20 cases had centromeric material from both chromosomes involved in the WAT, while 8 cases had centromeric material from only one chromosome detectable by FISH. The existence of balanced WATs as sole changes, the presence o f WATs in patients with biclonal disorders, and their frequent observation in complex karyotypes in patients with secondary disease suggest that, in addition to being pathognomic in some patients, they may also provide a general measure o f genomic instability associated with neoplastic development aud/or exposure to clastogenic/earcinogenic agents.
165
B7
CYTOGENETIC ANALYSIS OF A GLIOBLASTOMA IN A PATIENT WITH TURCOT SYNDROME. Roherty. SE, Richldnd, KE, Chilcote R. Genzyme Genetics, Santa Fe, N M (SER, KER); UCi, Irvine, C A (RC) Turcot syndrome is characterized by the concurrence o f colorectal tumors and primary brain tumors, with other tumor types also occurring. Familial cases have been associated with germliue mutations of the APC, hMI,HI or hPMS2 genes. The brain tumors found in these families are primarily medunoblastomas (APC gene mutations) or glioblastomas (HNPCC geue mutations). We describe the first cytogenetic study o f a glioblastoma associated with Tureot syndrome. The tumor was surgically reseeted from a 19-year-old female patient, who had a history o f intestinal polyps and, at age 14, had been diagnosed with fibrnsarcoma and treated with radiation and VP- 16. Cytogenetically the giioblastoma was characterized by clonal heterogeneity, with four pseudodiploid clones present. There was a preponderance of balanced structural rearrangements, with two o f the four clones demonstrating only balanced rearrangements. The other two clones had imbalances affecting chromosome 3, as well as a number of balanced translocations. Three of the four clones had jumping translocations involving 19q13. This tumor was very aggressive and recurred within six weeks, and the patient died soon after. The pattern of abnormalities observed in this tumor is unusual but is more similar to that reported for recurrent gliomas rather than for primary gliomas, w h i c h are more often aueuploid, pniyplnid and have unbalanced structural rearrangements. Whether this pattern is typical of glioblastomas associated with Turcot syndrome is unknown, but potential association between possible radiosensitivity, replication error repair mutations and cytogenetic abnormalities warrants further investigation.
B8
UNUSUAL KARYOTYPES IN TWO SYNOVIAL SARCOMAS. Roland. B. Departments o f Pathology and Medical Genetics, University o f Calgary, Calgary, Alberta, Canada. The characteristic chromosome abnormality in synovial sarcoma is t(X;18)(pl 1.2;ql 1.2), which is observed in over 80% o f abnormal karyotypod synovial sarcomas. Secondary chromosome abnormalities are frequent, with 75% of reported cases having structural aud/or numerical abnormalities in addition to t(X; 18). In this report, two synovial sarcomas with unusual karyotypes are described. The first case is a 22 cm primary thigh mass with spindle cell morphology from a 24 year old female. The tumour has a near-triploid karyotype o f 66-75,X,-X, t(X;18)(pll.2;qll.2),+2,+4,+5,-7,+9,+12,+14,-15,+22,+3mar[cp3]/46,XX[21. The second case is a primary lung mass with monomorphic spindle cell morphology, from a 46 year old female. The tumour kas a karyotype of 45,X,t(X;3; 18)(p l l . 2 ; q 2 9 ; q l 1.2),- 18113]. Clinical follow-up in both cases is less than 6 months. These two tumours are of interest because they have the typical U'anslocation for synovial sarcoma, hut also have unique chromosome abnormalities. T h e majority of cases of synovial sarcoma previously reported have been near diploid, with oniy three previous turnouts having chromosome counts above the hyperdiploid range. The tumunr presented here is the second in the near-triploid range. Several 3-way trauslocations involving chromosomes X, 18 and another autosome have been reported previously. The loss o f the normal chromosome 18 in the second case reported here, leaving only the tranalocated 18, is however, unique. Further tumour karyotypes with clinical correlation are required to determine the significance o f the near-triploid karyotype and the loss o f the normal chromosome 18.