891601 林子云 , 2002, Summer Tumor targeting and Prodrug Lab

Institute of Biomedical Sciences (IBMS) at the Academia Sinica in Taipei, TaiwanSteve Roffler

探討因前驅藥物療法所引發的防禦性免疫反應機制；利用人工分子演化的技術來增進免疫酵素的活性。亦試圖利用分子技術法表現單鏈單株抗體以及活化前驅藥物之酵素。 研究將融合蛋白表現在哺乳類細胞表面的基因療法，以應用在特殊的淋巴細胞調控上。同時探討融合蛋白中不同的區域對其運送和滯留於細胞表面的影響。探討細胞腫瘤轉移的機制 ，及調控內皮細胞血管新生的機身。目前著重於探討在癌細胞轉移與血管新生過程中 間的交互作用及其訊息傳遞路徑。

891601 林子云2002, Summer

Expression rate of CD13 and L6 on CL1-5 will be induced

by growth factors中研院生醫所Steve Roffler

L6 CD13

CL1-5

invasion

HUVECL6

CD13angiogenesis

CL1-5 GFs , Hypoxia

Purpose and introduction

We Study how the two surface proteins, CD13 and L6, are induced in different condition of medium. This might be helpful to illustrate roles of the two proteins in invasion of CL1-5.

Fluorescence-Activated Cell Sorter

Fluorescence-Activated Cell SorterTo define characteristics of unfamiliar cellsTo isolate specific cells for growth, cloning

or PCR Performing by the correlation between light

scatter patterns and cell properties such as DNA or RNA content, shape, size or texture.

•Scattered light and fluorescence signals are generated and the sort logic boards make a decision as to whether the cell is to be sorted or not.

• The cytometer waits until that cells has traveled from the intercept to the break-off point and then charges the stream. So as the drop containing the cell of interest leaves the solid fluid stream it will carry a charge, either positive or negative.

•The charged drop passes through two high voltage deflection plates and will be attracted towards the plate of opposite polarity.

•Dual-parameter plots of combinations of light scatter and fluorescence

•Forward light scatter: size

•Sideward light scatter: small cellular structures.

•The fluorescence:the amount of cellular pigment and its composition.

Cell type Condition

CL1-5

Versene treated

Light trypsin treated

Heavy trypsin treated

Recovery for short term after trypsin treated

Recovery for medium term after trypsin treated

Recovery for long term after trypsin treated

0. ProtocalsI. Test the better condition to harvest CL1-5

Methods

Run FACS, 1st abs=4B8, L6, and HB65, 2nd ab=Go Mo Ig(G+A+M)-FITCExpression level = FL1-intensity of 4B8 or L6 minus that of HB65.

0

100

200

300

400

verc

ene

Ligh

t tryp

sin

Hea

vy try

psin

reco

very

10m

in

reco

very

30m

in

reco

very

60m

in

CD

13 e

xpre

ssio

n

1 10 100 1000 10000FL1-H

0

100

200

300

# C

ell

s

V2+HB65V2+4B8T2+4B8T10+4B8R60+4B8R30+4B8R10+4B8

1 10 100 1000 10000FL1-H

# C

ell

s

0

100

200

300

V2+HB65V2+L6T2+L6T10+L6R60+L6R30+L6R10+L6

0

50

100

150

200

verc

ene

Ligh

t tryp

sin

Hea

vy try

psin

reco

very

10m

in

reco

very

30m

in

reco

very

60m

in

L6

expr

essi

on

Cell type Condition i Condition ii

HMEC-1

EBM-2+15%FBS+10µM L-glutamine+10nM EGFs+1µg/ml HDC

EBM-2+0%FBS

EBM-2+5%FBS EBM-2+VEGF

EBM-2+1%FBS EBM-2+TNF-

EBM-2+0%FBS

II. The influence of growth factors to CD13 expression on HMEC-1 cell

1. Starve the cells for 24 hr.2. Culture cells in the medium above for another 36 hr.3. Harvest cells by versene**. Run FACS 1st atbs=4B8, L6, and HB65, 2nd atb=Go Mo Ig(G+A+M) Expression rate = FL1-intensity of 4B8 or L6 minus that of HB65.

III. the influence of growth factors to CD13 expression on CL1-5 cell

Cell type Condition i Condition ii

CL1-5

RPMI+10%BCS RPMI+0%BCS

RPMI+5%BCS RPMI+VEGF

RPMI+1%BCS RPMI+TNF-RPMI+0%BCS

1. Starve the cells for 24 hr.2. Culture cells in the medium above for another 36 hr.3. Harvest cells by versene**. Run FACS 1st atbs=4B8, L6, and HB65, 2nd atb=Go Mo Ig(G+A+M) Expression rate = FL1-intensity of 4B8 or L6 minus that of HB65.

0

10

20

30

40

50

NONE

VEGF

TNF-

CoCl

2

CD

13 e

xpre

ssio

n r

ate

condition

80

0

20

40

60

NONE

VEGF

TNF-

CoCl2

L6

exp

ress

ion

rat

e

condition

HMEC-1 with growth factors

Result

0

50

100

150

200

250

10%B

CS

5%BCS

1%BCS

0%BCS

condition

CD

13 e

xpre

ssio

n

0

100

200

300

400

500

10%B

CS

5%BCS

1%BCS

0%BCS

L6

exp

ress

ion

condition

CL1-5 serum starvation

CL1-5 with growth factors

0

50

100

150

200

250

300

NONE

VEGF

TNF-

CoCl2

CD

13 e

xpre

ssio

n r

ate

condition1 10 100 1000 10000

FL1-H

0

100

200

300

# C

ell

s

4B8+CoCl24B8+None4B8+TNF-4B8+VEGF

# C

ell

s

1 10 100 1000 10000

FL1-H

0

50

100

150

200

250

L6+CoCl2L6+NoneL6+TNF-L6+VEGF

0

50

100

150

200

250

NONE

VEGF

TNF-

CoCl

2

L6

exp

ress

ion

rat

e

condition

Conclusion

•L6 expressed on HMEC-1 might be induced by some growth factors, including TNF-.• CD13 and L6 expressed on CL1-5 might both be induced by growth factors. •According to the similar trends of CD13 and L6 expression levels on CL1-5, the growth factors might alter the whole CD13-L6 complex expression level. And the invasion of CL1- 5 might be relative to CD13 as well as L6 since they can be induced at the same % of serum.

Future works

Find out the real functions of CD13 and L6 in the process of invasion in CL1-5 by other methods such as genetics.

Test if CD13 and L6 have anything to do with vascularization of HMEC-1 and angiogenesis in tumor formation by other methods.

Immunoenzyme and prodrug treatment of rat malignant ascites effectively controls disseminated tumor growth.