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Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function.

Yu Q, Toole BP, Stamenkovic I - J. Exp. Med. (1997)

Bottom Line: To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro.However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis.Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology Unit and MGH Cancer Center, Massachusetts General Hospital, Charlestown Navy Yard, Boston, Massachusetts 02129, USA.

ABSTRACT
To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro. Local release of soluble CD44 by the transfectants inhibited the ability of endogenous cell surface CD44 to bind and internalize hyaluronan and to mediate TA3 cell invasion of hyaluronan-producing cell monolayers. Mice intravenously injected with parental TA3/St cells developed massive pulmonary metastases within 21-28 d, whereas animals injected with TA3sCD44 cells developed few or no tumors. Tracing of labeled parental and transfectant tumor cells revealed that both cell types initially adhered to pulmonary endothelium and penetrated the interstitial stroma. However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis. Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells. These observations provide direct evidence that cell surface CD44 function promotes tumor cell survival in invaded tissue and that its suppression can induce apoptosis of the invading tumor cells, possibly as a result of impairing their ability to penetrate the host tissue hyaluronan barrier.

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(A) Expression of soluble CD44 prevents lung metastasis of  TA3 cells. Lungs from two representative mice injected with the  TA3sCD44v6-10 No. 17 cells reveal no apparent tumor nodules (a and  b); lungs from two representative mice injected with TA3neo No. 1 display innumerable tumor nodules throughout the lung parenchyma (c and  d). (B) Tumors derived from lungs of mice injected with TA3sCD44v6-v10 No. 12 cells have lost soluble CD44 expression. Five independent tumor nodules were dissected out from the lung, and RT-PCR was performed using total RNA derived from each nodule (lanes 2–11). The  primers used were: lanes 2, 4, 6, 8, 10, and 14, primer 1f and primer 20r,  to detect expression of endogenous CD44; lanes 3, 5, 7, 9, 11, and 15,  primer 1f and new v10r to detect expression of soluble CD44 (16); lanes  12–15: total RNA from cultured TA3sCD44v6-v10 No. 12 cells was  used as template for lanes 12 and 13, negative controls, where PCR was  performed with 1f primers only; lanes 14 and 15, positive controls where  PCR was performed with primers 1f and 20r (lane 14), and 1f and new  v10r (lane 15). Molecular weight markers (100-bp ladder) are shown in  lane 1. RT-PCR analysis indicates that all five tumor nodules express endogenous but not soluble recombinant CD44.
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Figure 2: (A) Expression of soluble CD44 prevents lung metastasis of TA3 cells. Lungs from two representative mice injected with the TA3sCD44v6-10 No. 17 cells reveal no apparent tumor nodules (a and b); lungs from two representative mice injected with TA3neo No. 1 display innumerable tumor nodules throughout the lung parenchyma (c and d). (B) Tumors derived from lungs of mice injected with TA3sCD44v6-v10 No. 12 cells have lost soluble CD44 expression. Five independent tumor nodules were dissected out from the lung, and RT-PCR was performed using total RNA derived from each nodule (lanes 2–11). The primers used were: lanes 2, 4, 6, 8, 10, and 14, primer 1f and primer 20r, to detect expression of endogenous CD44; lanes 3, 5, 7, 9, 11, and 15, primer 1f and new v10r to detect expression of soluble CD44 (16); lanes 12–15: total RNA from cultured TA3sCD44v6-v10 No. 12 cells was used as template for lanes 12 and 13, negative controls, where PCR was performed with 1f primers only; lanes 14 and 15, positive controls where PCR was performed with primers 1f and 20r (lane 14), and 1f and new v10r (lane 15). Molecular weight markers (100-bp ladder) are shown in lane 1. RT-PCR analysis indicates that all five tumor nodules express endogenous but not soluble recombinant CD44.

Mentions: Each of the six transfectants was injected into the tail vein of nude (nu/nu) or A/jax mice in three independent experiments. In the first and second experiments, three nude and three A/jax mice were injected with 1 × 106 cells of each transfectant. In the third experiment, a minimum of six A/jax mice were injected with each transfectant (summarized in Table 2). 3 wk after injection, mice which had received TA3neo No. 1 and No. 8 displayed severe weight loss, and they were killed 1 wk later. However, animals which had received TA3sCD44v6-10 and v8-10 cells showed no signs of distress even several months after tumor cell injection. The mice injected with each TA3sCD44 transfectant were killed at the end of the fourth week and compared for metastatic tumor growth with that in TA3neo mice. All of the mice injected with TA3neo No. 1 and No. 8 displayed massive pulmonary metastases (Fig. 2 A, c and d). By contrast, mice injected with TA3sCD44s transfectants displayed either no tumor nodules (TA3sCD44v6-10 No. 17 and TA3sCD44v8-10 No. 13) or only a small number (<30) of tumor nodules per lung (TA3sCD33v6-10 No. 12 and TA3sCD44v8-10 No. 19, Fig. 2 A, a and b, and Table 2). No tumor nodules were found in other organs. RT-PCR on total RNA derived from several of the scarce tumor nodules from the lungs of mice injected with TA3sCD44v6-10 No. 12 revealed that these tumors had lost expression of transfected soluble CD44 while retaining endogenous cell surface CD44 expression (Fig. 2 B). This result provides an internal control that underscores the notion that local release of soluble, tumor-derived CD44 inhibits formation of lung metastasis by TA3 cells.


Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function.

Yu Q, Toole BP, Stamenkovic I - J. Exp. Med. (1997)

(A) Expression of soluble CD44 prevents lung metastasis of  TA3 cells. Lungs from two representative mice injected with the  TA3sCD44v6-10 No. 17 cells reveal no apparent tumor nodules (a and  b); lungs from two representative mice injected with TA3neo No. 1 display innumerable tumor nodules throughout the lung parenchyma (c and  d). (B) Tumors derived from lungs of mice injected with TA3sCD44v6-v10 No. 12 cells have lost soluble CD44 expression. Five independent tumor nodules were dissected out from the lung, and RT-PCR was performed using total RNA derived from each nodule (lanes 2–11). The  primers used were: lanes 2, 4, 6, 8, 10, and 14, primer 1f and primer 20r,  to detect expression of endogenous CD44; lanes 3, 5, 7, 9, 11, and 15,  primer 1f and new v10r to detect expression of soluble CD44 (16); lanes  12–15: total RNA from cultured TA3sCD44v6-v10 No. 12 cells was  used as template for lanes 12 and 13, negative controls, where PCR was  performed with 1f primers only; lanes 14 and 15, positive controls where  PCR was performed with primers 1f and 20r (lane 14), and 1f and new  v10r (lane 15). Molecular weight markers (100-bp ladder) are shown in  lane 1. RT-PCR analysis indicates that all five tumor nodules express endogenous but not soluble recombinant CD44.
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Figure 2: (A) Expression of soluble CD44 prevents lung metastasis of TA3 cells. Lungs from two representative mice injected with the TA3sCD44v6-10 No. 17 cells reveal no apparent tumor nodules (a and b); lungs from two representative mice injected with TA3neo No. 1 display innumerable tumor nodules throughout the lung parenchyma (c and d). (B) Tumors derived from lungs of mice injected with TA3sCD44v6-v10 No. 12 cells have lost soluble CD44 expression. Five independent tumor nodules were dissected out from the lung, and RT-PCR was performed using total RNA derived from each nodule (lanes 2–11). The primers used were: lanes 2, 4, 6, 8, 10, and 14, primer 1f and primer 20r, to detect expression of endogenous CD44; lanes 3, 5, 7, 9, 11, and 15, primer 1f and new v10r to detect expression of soluble CD44 (16); lanes 12–15: total RNA from cultured TA3sCD44v6-v10 No. 12 cells was used as template for lanes 12 and 13, negative controls, where PCR was performed with 1f primers only; lanes 14 and 15, positive controls where PCR was performed with primers 1f and 20r (lane 14), and 1f and new v10r (lane 15). Molecular weight markers (100-bp ladder) are shown in lane 1. RT-PCR analysis indicates that all five tumor nodules express endogenous but not soluble recombinant CD44.
Mentions: Each of the six transfectants was injected into the tail vein of nude (nu/nu) or A/jax mice in three independent experiments. In the first and second experiments, three nude and three A/jax mice were injected with 1 × 106 cells of each transfectant. In the third experiment, a minimum of six A/jax mice were injected with each transfectant (summarized in Table 2). 3 wk after injection, mice which had received TA3neo No. 1 and No. 8 displayed severe weight loss, and they were killed 1 wk later. However, animals which had received TA3sCD44v6-10 and v8-10 cells showed no signs of distress even several months after tumor cell injection. The mice injected with each TA3sCD44 transfectant were killed at the end of the fourth week and compared for metastatic tumor growth with that in TA3neo mice. All of the mice injected with TA3neo No. 1 and No. 8 displayed massive pulmonary metastases (Fig. 2 A, c and d). By contrast, mice injected with TA3sCD44s transfectants displayed either no tumor nodules (TA3sCD44v6-10 No. 17 and TA3sCD44v8-10 No. 13) or only a small number (<30) of tumor nodules per lung (TA3sCD33v6-10 No. 12 and TA3sCD44v8-10 No. 19, Fig. 2 A, a and b, and Table 2). No tumor nodules were found in other organs. RT-PCR on total RNA derived from several of the scarce tumor nodules from the lungs of mice injected with TA3sCD44v6-10 No. 12 revealed that these tumors had lost expression of transfected soluble CD44 while retaining endogenous cell surface CD44 expression (Fig. 2 B). This result provides an internal control that underscores the notion that local release of soluble, tumor-derived CD44 inhibits formation of lung metastasis by TA3 cells.

Bottom Line: To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro.However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis.Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology Unit and MGH Cancer Center, Massachusetts General Hospital, Charlestown Navy Yard, Boston, Massachusetts 02129, USA.

ABSTRACT
To understand how the hyaluronan receptor CD44 regulates tumor metastasis, the murine mammary carcinoma TA3/St, which constitutively expresses cell surface CD44, was transfected with cDNAs encoding soluble isoforms of CD44 and the transfectants (TA3sCD44) were compared with parental cells (transfected with expression vector only) for growth in vivo and in vitro. Local release of soluble CD44 by the transfectants inhibited the ability of endogenous cell surface CD44 to bind and internalize hyaluronan and to mediate TA3 cell invasion of hyaluronan-producing cell monolayers. Mice intravenously injected with parental TA3/St cells developed massive pulmonary metastases within 21-28 d, whereas animals injected with TA3sCD44 cells developed few or no tumors. Tracing of labeled parental and transfectant tumor cells revealed that both cell types initially adhered to pulmonary endothelium and penetrated the interstitial stroma. However, although parental cells were dividing and forming clusters within lung tissue 48 h following injection, >80% of TA3sCD44 cells underwent apoptosis. Although sCD44 transfectants displayed a marked reduction in their ability to internalize and degrade hyaluronan, they elicited abundant local hyaluronan production within invaded lung tissue, comparable to that induced by parental cells. These observations provide direct evidence that cell surface CD44 function promotes tumor cell survival in invaded tissue and that its suppression can induce apoptosis of the invading tumor cells, possibly as a result of impairing their ability to penetrate the host tissue hyaluronan barrier.

Show MeSH
Related in: MedlinePlus