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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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Binding of HA and CD44 expression by TA3/St cells. (A) Binding of FL-HA by TA3/ST cells as assessed by FACS® analysis (a). The ability  of TA3/St cells to bind FL-HA completely abrogated by the blocking anti-CD44 monoclonal antibody KM201 (b), whereas control anti–ICAM-1 mAb  has no effect (c). (B) Expression of multiple CD44 isoforms by TA3/St cells: RT-PCR was performed using the oligonucleotide primers listed in Materials  and Methods and total RNA from TA3/St cells, and the products were analyzed on 1% agarose gels. Lane 1, 100-bp DNA reference ladder (GIBCO  BRL); lane 2, PCR products using a forward primer corresponding to exon 5 (5f) and a reverse primer corresponding to exon 16 (16r). A 120-bp product representing the expression of the standard CD44 isoform containing no variant exons (CD44H) is indicated (white arrow); the presence of larger  products indicates expression of multiple CD44 variants. Lanes 3–12, the reverse 16r primer was used together with forward primers v1f through v10f.  The products in these lanes demonstrate that TA3/St cells express a range of CD44 variants containing variant exons v1 through v10. (C) Western blot  analysis of CD44 expression in lysates (a) and supernatants (b) of parental and soluble CD44-transfected TA3/St cells. Lysates in a and supernatants in b  were from: lane 1, TA3neo No. 1; lane 2, TA3neo No. 8; lane 3, TA3sCD44 v8-v10 No. 13; lane 4, TA3sCD44v8-10 No. 19; lane 5, TA3sCD44v6-v10 No. 12; lane 6, TA3sCD44v6-10 No. 17 cells. TA3neo No. 1 and No. 8 cells express CD44H (∼80 kD), and several larger CD44 isoforms; lysates  from TA3 cells expressing soluble CD44 show a similar pattern of CD44 isoform expression, except that the abundance of CD44 proteins of ∼80 Kd,  which corresponds to the Mr of soluble CD44 isoforms, is increased as expected. Western blot analysis of TA3/St cell culture supernatants (b) reveals that  TA3neo No. 1 and No. 8 cells (lanes 1 and 2) do not produce soluble CD44, whereas TA3sCD44 transfectants produce variable amounts of soluble  CD44 (lanes 3–6). Arrows indicate molecular weight markers, which are, from top to bottom, 203, 118, 86, and 52 kD.
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Figure 1: Binding of HA and CD44 expression by TA3/St cells. (A) Binding of FL-HA by TA3/ST cells as assessed by FACS® analysis (a). The ability of TA3/St cells to bind FL-HA completely abrogated by the blocking anti-CD44 monoclonal antibody KM201 (b), whereas control anti–ICAM-1 mAb has no effect (c). (B) Expression of multiple CD44 isoforms by TA3/St cells: RT-PCR was performed using the oligonucleotide primers listed in Materials and Methods and total RNA from TA3/St cells, and the products were analyzed on 1% agarose gels. Lane 1, 100-bp DNA reference ladder (GIBCO BRL); lane 2, PCR products using a forward primer corresponding to exon 5 (5f) and a reverse primer corresponding to exon 16 (16r). A 120-bp product representing the expression of the standard CD44 isoform containing no variant exons (CD44H) is indicated (white arrow); the presence of larger products indicates expression of multiple CD44 variants. Lanes 3–12, the reverse 16r primer was used together with forward primers v1f through v10f. The products in these lanes demonstrate that TA3/St cells express a range of CD44 variants containing variant exons v1 through v10. (C) Western blot analysis of CD44 expression in lysates (a) and supernatants (b) of parental and soluble CD44-transfected TA3/St cells. Lysates in a and supernatants in b were from: lane 1, TA3neo No. 1; lane 2, TA3neo No. 8; lane 3, TA3sCD44 v8-v10 No. 13; lane 4, TA3sCD44v8-10 No. 19; lane 5, TA3sCD44v6-v10 No. 12; lane 6, TA3sCD44v6-10 No. 17 cells. TA3neo No. 1 and No. 8 cells express CD44H (∼80 kD), and several larger CD44 isoforms; lysates from TA3 cells expressing soluble CD44 show a similar pattern of CD44 isoform expression, except that the abundance of CD44 proteins of ∼80 Kd, which corresponds to the Mr of soluble CD44 isoforms, is increased as expected. Western blot analysis of TA3/St cell culture supernatants (b) reveals that TA3neo No. 1 and No. 8 cells (lanes 1 and 2) do not produce soluble CD44, whereas TA3sCD44 transfectants produce variable amounts of soluble CD44 (lanes 3–6). Arrows indicate molecular weight markers, which are, from top to bottom, 203, 118, 86, and 52 kD.

Mentions: To investigate how tumor cell surface CD44 expression influences the metastatatic process, we expressed soluble, truncated CD44 isoforms, composed of the extracellular domain containing different combinations of variant exons but lacking the transmembrane and intracellular domains, in the mouse mammary carcinoma TA3/St. TA3/St cells constitutively express several cell surface CD44 isoforms ranging from 80 to 220 kD (Fig. 1, B and C), display CD44-mediated binding of HA (Fig. 1 A), and rapidly form tumors in the lung after intravenous injection. We reasoned that the soluble CD44 isoforms should compete with and suppress or abrogate the normal function of endogenous cell surface CD44, thereby providing insight into how CD44 expression regulates TA3/St cell behavior in vivo. Naturally occurring soluble truncated CD44 receptors, which bear a stop codon in exon v10, have recently been identified in G8 mouse fetal myoblasts (24). Two soluble truncated CD44 isoforms containing variable exons v6–v10 and v8–v10 were isolated from G8 cell RNA by RT-PCR, introduced into the pCR 3-Uni eukaryotic expression vector (Invitrogen Corp.) and stably expressed in TA3/St cells. Four independent TA3/St transfectants expressing two different soluble CD44 isoforms (termed sCD44v6-10 No. 12 and No. 17 and sCD44v8-10 No. 13 and No. 19) and two independent clones transfected with the expression vector alone (neo No. 1 and No. 8) were selected for further study. All six transfectants expressed comparable levels of cell surface CD44, as assessed by FACS® analysis, and displayed a similar baseline proliferation rate as measured by [3H]thymidine incorporation in vitro (Table 1). Western blot analysis of transfectant supernatants using the mAb KM201, which recognizes an epitope common to all CD44 isoforms in the NH2 domain, revealed that sCD44v6-10 and sCD44v8-10 transfectants, but not neotransfectants, secreted soluble CD44 into their culture medium (Fig. 1 C, b).


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)

Binding of HA and CD44 expression by TA3/St cells. (A) Binding of FL-HA by TA3/ST cells as assessed by FACS® analysis (a). The ability  of TA3/St cells to bind FL-HA completely abrogated by the blocking anti-CD44 monoclonal antibody KM201 (b), whereas control anti–ICAM-1 mAb  has no effect (c). (B) Expression of multiple CD44 isoforms by TA3/St cells: RT-PCR was performed using the oligonucleotide primers listed in Materials  and Methods and total RNA from TA3/St cells, and the products were analyzed on 1% agarose gels. Lane 1, 100-bp DNA reference ladder (GIBCO  BRL); lane 2, PCR products using a forward primer corresponding to exon 5 (5f) and a reverse primer corresponding to exon 16 (16r). A 120-bp product representing the expression of the standard CD44 isoform containing no variant exons (CD44H) is indicated (white arrow); the presence of larger  products indicates expression of multiple CD44 variants. Lanes 3–12, the reverse 16r primer was used together with forward primers v1f through v10f.  The products in these lanes demonstrate that TA3/St cells express a range of CD44 variants containing variant exons v1 through v10. (C) Western blot  analysis of CD44 expression in lysates (a) and supernatants (b) of parental and soluble CD44-transfected TA3/St cells. Lysates in a and supernatants in b  were from: lane 1, TA3neo No. 1; lane 2, TA3neo No. 8; lane 3, TA3sCD44 v8-v10 No. 13; lane 4, TA3sCD44v8-10 No. 19; lane 5, TA3sCD44v6-v10 No. 12; lane 6, TA3sCD44v6-10 No. 17 cells. TA3neo No. 1 and No. 8 cells express CD44H (∼80 kD), and several larger CD44 isoforms; lysates  from TA3 cells expressing soluble CD44 show a similar pattern of CD44 isoform expression, except that the abundance of CD44 proteins of ∼80 Kd,  which corresponds to the Mr of soluble CD44 isoforms, is increased as expected. Western blot analysis of TA3/St cell culture supernatants (b) reveals that  TA3neo No. 1 and No. 8 cells (lanes 1 and 2) do not produce soluble CD44, whereas TA3sCD44 transfectants produce variable amounts of soluble  CD44 (lanes 3–6). Arrows indicate molecular weight markers, which are, from top to bottom, 203, 118, 86, and 52 kD.
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Figure 1: Binding of HA and CD44 expression by TA3/St cells. (A) Binding of FL-HA by TA3/ST cells as assessed by FACS® analysis (a). The ability of TA3/St cells to bind FL-HA completely abrogated by the blocking anti-CD44 monoclonal antibody KM201 (b), whereas control anti–ICAM-1 mAb has no effect (c). (B) Expression of multiple CD44 isoforms by TA3/St cells: RT-PCR was performed using the oligonucleotide primers listed in Materials and Methods and total RNA from TA3/St cells, and the products were analyzed on 1% agarose gels. Lane 1, 100-bp DNA reference ladder (GIBCO BRL); lane 2, PCR products using a forward primer corresponding to exon 5 (5f) and a reverse primer corresponding to exon 16 (16r). A 120-bp product representing the expression of the standard CD44 isoform containing no variant exons (CD44H) is indicated (white arrow); the presence of larger products indicates expression of multiple CD44 variants. Lanes 3–12, the reverse 16r primer was used together with forward primers v1f through v10f. The products in these lanes demonstrate that TA3/St cells express a range of CD44 variants containing variant exons v1 through v10. (C) Western blot analysis of CD44 expression in lysates (a) and supernatants (b) of parental and soluble CD44-transfected TA3/St cells. Lysates in a and supernatants in b were from: lane 1, TA3neo No. 1; lane 2, TA3neo No. 8; lane 3, TA3sCD44 v8-v10 No. 13; lane 4, TA3sCD44v8-10 No. 19; lane 5, TA3sCD44v6-v10 No. 12; lane 6, TA3sCD44v6-10 No. 17 cells. TA3neo No. 1 and No. 8 cells express CD44H (∼80 kD), and several larger CD44 isoforms; lysates from TA3 cells expressing soluble CD44 show a similar pattern of CD44 isoform expression, except that the abundance of CD44 proteins of ∼80 Kd, which corresponds to the Mr of soluble CD44 isoforms, is increased as expected. Western blot analysis of TA3/St cell culture supernatants (b) reveals that TA3neo No. 1 and No. 8 cells (lanes 1 and 2) do not produce soluble CD44, whereas TA3sCD44 transfectants produce variable amounts of soluble CD44 (lanes 3–6). Arrows indicate molecular weight markers, which are, from top to bottom, 203, 118, 86, and 52 kD.
Mentions: To investigate how tumor cell surface CD44 expression influences the metastatatic process, we expressed soluble, truncated CD44 isoforms, composed of the extracellular domain containing different combinations of variant exons but lacking the transmembrane and intracellular domains, in the mouse mammary carcinoma TA3/St. TA3/St cells constitutively express several cell surface CD44 isoforms ranging from 80 to 220 kD (Fig. 1, B and C), display CD44-mediated binding of HA (Fig. 1 A), and rapidly form tumors in the lung after intravenous injection. We reasoned that the soluble CD44 isoforms should compete with and suppress or abrogate the normal function of endogenous cell surface CD44, thereby providing insight into how CD44 expression regulates TA3/St cell behavior in vivo. Naturally occurring soluble truncated CD44 receptors, which bear a stop codon in exon v10, have recently been identified in G8 mouse fetal myoblasts (24). Two soluble truncated CD44 isoforms containing variable exons v6–v10 and v8–v10 were isolated from G8 cell RNA by RT-PCR, introduced into the pCR 3-Uni eukaryotic expression vector (Invitrogen Corp.) and stably expressed in TA3/St cells. Four independent TA3/St transfectants expressing two different soluble CD44 isoforms (termed sCD44v6-10 No. 12 and No. 17 and sCD44v8-10 No. 13 and No. 19) and two independent clones transfected with the expression vector alone (neo No. 1 and No. 8) were selected for further study. All six transfectants expressed comparable levels of cell surface CD44, as assessed by FACS® analysis, and displayed a similar baseline proliferation rate as measured by [3H]thymidine incorporation in vitro (Table 1). Western blot analysis of transfectant supernatants using the mAb KM201, which recognizes an epitope common to all CD44 isoforms in the NH2 domain, revealed that sCD44v6-10 and sCD44v8-10 transfectants, but not neotransfectants, secreted soluble CD44 into their culture medium (Fig. 1 C, b).

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