Indian Journal o f Cllnlcal Blochemlstry 1992, 7(2) 81-88

I N T R A C E L L ~ P R O T E I N S A S T U M O R M A R K E R S

ALPANA GUPTA § T MALATI* AND P D GUPTA* Department of Biochemistry, Nizams Ins t i tu te of Medical Sciences, Hyde rabad - 500 482. India *Centre for Cellular & Molecular Biology. Hyderabad - 500 007. India.

INTRODUCTION

During m a l i g n a n t t r ans fo rma t ion of cells, there is a chaos in the p r o g r a m m e d gene expression. Cer ta in cells wh ich are fully differentiated s u c h as hepa tocy tes in wh ich a l b u m i n is

the marke r protein, s t a r t p roduc ing AFP-and oncofetal ant igen, w h e n they become cance rous . In adult subjects , if one detects AFP in the serum, there is a chance t h a t the indiv idual m a y be

suffering f rom hepa toce l lu la r ca rc inoma ( 1,2). Some of the gene produc ts which are not expressed

or expressed in very low a m o u n t s in normal hea l thy cells, m a y be overexpressed af ter m a l i g n a n t

t ransformat ion . S u c h s u b s t a n c e s are secreted in the s e r u m a n d c a n be u s e d to d iagnose

malignancies . Thus , t u m o r marke r s c a n be defined as a biochemical s u b s t a n c e p roduced by the

tumor which w h e n p resen t in s ignif icant detectable a m o u n t s , indicates the p resence of a cance r

(3-6). Essent ia l ly , a n y molecular species produced in a b n o r m a l (low or high) a m o u n t s or u n d e r

abnorma l c i r c u m s t a n c e s m a y become useful as a diagnost ic tool. Biochemically, t u m o r marke r s

are u sua l l y con juga ted prote ins which may be p resen t in m inu t e quant i t i es in the n o r m a l h e a l t h y individual, b u t the elevated levels of these subs t ances indicate mal ignanc ies .

Excel lent reviews are available on secretory proteins which ac t as t u m o r marke r s (7,8),

however, the u se of in t racel lu lar proteins as cancer markers is no t in vogue, therefore, very little

in format ion is available a b o u t them. The scope of this review is to provide wha tever little in format ion available a b o u t intracel lular proteins which can be used as t u m o r marke r s a n d men t ion the i r appl ica t ions in t u m o r biology.

A p p l i c a t i o n s o f t u m o r markers

Tumor marke r s have a diverse set of applicat ions right from de tec t ion of cance r s to the i r therapy. Impor t an t appl icat ions include their ability to provide valuable in fo rmat ion regard ing the

s tag ing of tumors . Markers can also be used to detect me tas t a s i s by rad io imrnuno m e t h o d s (9).

Tumor m a r k e r s he lp in prognosis or t racking the behaviour o f a tumor . The m a r k e r levels, h igh or low, indica te whe the r a t u m o r is progressing or regressing respectively. According to the t u m o r

type a n d its behaviour , a n appropr ia te method of the rapy can be decided upon , e i ther chemo-

therapy, rad io therapy , resect ion of the t u m o r or a combina t ion of a n y of these me thods . The 11kelLhood of r e sponse to the mode of t r e a tmen t c a n also be predic ted by eva lua t ion of the t u m o r

m a r k e r (8). The m o s t frui tful applicat ion o f t umor markers is monitoring the t u m o r act ivi ty a n d

efficacy of t r ea tmen t . This has been realized most cons is tent ly in ges ta t iona l t rophoblas t ic t u m o r s

(chor iocarcinomas) in w o m e n (10) and n o n s e m u n o m a t o u s germ cell t u m o r s in m e n (11 ). Besides

the above m e n t i o n e d applicat ions, the concent ra t ion of a t u m o r m a r k e r reflects a a dynamic

ba lance represen t ing the combina t ion of t u m o r activity and m a r k e r turnover . However, there is

A d d r e ~ for C o m m u n i c a t i o n Dr P D G u p t a Cent re for Cellular & Molecular Biology, Telephone : 00-91-842-8~2241 Telex : 0425-7046 CCMB IN, Fax : 00-91-842-851195

82 GUPTA ET AL. 1992

no t a un iversa l correla t ion between t u n m o r size and marke r concen t ra t ion . The u l t imate

appl ica t ion of t u m o r marke r s is to target an t ibody bound cytotoxic agen t s or the pa t ien t ' s own

i m m u n e cells to preferential ly control or des t roy mal ignan t cells while min imiz ing d a m a g e to

no rma l cells. In o ther words, the concept of i m m u n o t h e r a p y came t h r o u g h t u m o r marke r s in cance r m a n a g e m e n t .

Classification of Tumor markers

Depending on whe the r the marke r protein is secreted by the t u m o r cells or not , t u m o r

marke r s can be classified into two ma in types (12):

( I ) Tumor derived products ; and

(2) Tumor assoc ia ted products .

Tumor derived p roduc t s or molecules produced by the t umors are fu r the r subdiv ided into

two types:

(a) Syn thes ized products ; and

(b) Metabolical ly active subs t ances .

Synthes ized products may be:

(i) Oncofetal antigens: markers like (x-fetoprotein (5,13) a n d Carc inoembryon ic an t igen

(6,14) belong to this class. They are glycoproteins syn thes ized in the e m b r y o n a l s tage wi th a

m i n i m u m value reach ing a t adu l thood . Only in cer ta in specific mal ignancies , the i r level in the s e r u m is increased signif icant ly in adul ts .

(II) Ectopic products : The modified metabol i sm of t u m o r cells resu l t ing from increased cell

prol i ferat ion leads to increased syn thes i s of various enzymes w h e n compared to n o r m a l cells.

Marked increase in the activity of some enzymes for glycolysis, b iosyn thes i s of nucleic acid a n d

prote ins are charac ter i s t ic of a ma l ignan t metabolic process. As a resu l t of low specifici ty however, the i r d iagnost ic value for t u m o r diagnosis is limited.

(iii) Oncoplacental antigens : H u m a n chorionic gonadot ropin a n d p r e g n a n c y specific B-1 glycoprotein (SPI) are bes t k n o w n members of this series ( 15-17).

(2) Tumor associated products : These are factors accompany ing the m a l i g n a n t p h e n o m e n o n .

Al though this c lass of marke r s have low specificity, however, as addi t ional facul ta t ive marke r s they

c a n indicate ma l ignan t d iseases a n d provide addi t ional in format ion du r ing t ime course moni tor-

ing. This c lass inc ludes quant i ta t ive ly al tered s e r u m proteins s u c h as ferri t in a n d B 2 microglobul in (18-20) a n d in t race l lu lar proteins s u c h as cytoskeletal proteins (21-23) enzymes (kinases, phospha t a se s ) which are involved in phosphory la t ion of regulatory proteins (24,25), enzymes s u c h

as t r a n s g l u t a m i n a s e (26) which help in protein t r a n s a m i n a t i o n (cross- l inking of proteins) a n d

glycosyla t ion (27). Src gene family oncogene products can also be inc luded as in t rac l lu la r prote in t u m o r marke r s (28).

Specificity of tumor markers

Some t u m o r marke r s are believed to be specific for cer ta in ca rc inomas because they are

p roduced by those t umor s a n d hence are used to diagnose t ha t par t i cu la r mal ignancy . Table 1

below out l ines some of the t u m o r markers and the cancers for which they are specific (8).

VOL 7(2} 83 IntraceIlular Proteins as Tumor Markers

Ta bl e - 1

Tumor markers Type o f t u m o r for w h i c h s p e c i f i c

Carinoembryonic an t igen (CEA)

Alphafetoprotein {AFP)

~-Human chorionic

gonadotropin (~-HCG)

Prostate specific an t igen (PSA} and Prostate alkal ine p h o s p h a t a s e (PAP)

Calcitonin

Carcinoma an t igen 19-9 (CA 19-9)

Carcinoma an t igen 125 (CA 125) ~2- microglobulin

Creatine k inase (CK} CK-BB

Neuron specific enolase (NSE)

Lacatate dehyd rogenase (LDH)

Arginine vasopress in (AVP), Neurophysin,

Parathyroid ho rmone (PTH), Bombes in

Glycosyl t r ans fe rases

Pancreatic oncofetal an t igen {POA)

Tissue polypept ide an t igen

S o m e a n o m a l i e s

Well differentiated t umor s of the large bowel.

H e p a t o c e l l u l a r c a r c i n o m a s a n d n o n -

s e m i n o m a t o u s germ cell t umors . Chor iocarc inomas

Prostate cancer

Medullary thyroid ca r c inoma Epithelial tumors of the pancreas , co lorec tum

and s t omach

Non-mucinous ovar ian t u m o r s Multiple myelomas

Small cell lung cancer (SCLC}, b reas t a n d

prostate

Neuroendocr ine t u m o r s inc lud ing SCLC

Germ cell tes t icular t u m o r s

Lung

Ova .ry, breast , pancreas Pancreas

Bre,: ~ celor.,, lung

The above listed markers have been found to be elevated in the par t i cu la r ca rc inomas

ment ioned. The problem in diagnosis is tha t nonmal ignan t diseases c a n also be a s soc ia t ed with

the s ame m a r k e r abnormal i t ies . A marker can also be elevated in more t h a n one type of mal ignancy ,

making a diagnosis based solely on the marker a s s a y not reliable. For example, h igh C E A levels may indicate the presence of ei ther gas t ro intes t inal t rac t cancer, lung cancer , cance r o f the female genital t rac t or head a n d neck cancers (29). Another d rawback of de tec t ing cancers by t u m o r

marker a s s ays is low sensit ivi ty of the assay, which may be high for advanced or me tas t a t i c cance r but u s u a l l y is less t h a n 50% for early or localized cancer.

Epithe l ia l t u m o r markers

Recently, by us ing diagnost ic h is topathology and immunoh i s tochemis t ry , molecu la r com-

ponents t h a t are specifically expressed in epithelial cells have been recognised. These specific

componen t s have acqui red great importance in tumor biology and pathology a n d have been

designated as "epithelial differentiat ion markers". Such markers have two m a i n appl icat ions:

(a) in d i s t ingu i sh ing epithelial f rom non-epi thel ial tumors ; and

(b) in d i s t ingu i sh ing the type of epithelial tumor .

Impor tance is now being a t t ached to epithelial differentiat ion marke r s s u c h as cancer

associated an t igen [19-9] (30-32} and cancer associa ted an t igen [125] (32-36), epi thel ial mem-

84 GUPTA ET AL. 1992

b rane an t igen [EMA] (37), t i ssue polypeptide ant igen [TPA] (38), kera t ins (39-48), s q u a m o u s cell c a r c inoma an t igen [SCCA] (49-53) a n d Neuron specific enolase [NSE] (54-56).

In con t r a s t to o ther epithelial markers described above, kera t in f i laments are u su a l l y

un i formly d i s t r ibu ted a m o n g ca rc inoma cells and also, the degree of s tabi l i ty of ke ra t in express ion

in t u m o r s is r emarkab ly high. Therefore, kera t in is also a reliable marke r for (a) undi f fe ren t ia ted

and anap las t i c ca rc inomas , (b) disparate ly growing in f l t r a t ing ca rc inoma cells a n d (c) for me tas tas i z ing single ca rc inoma cells in suspens ion . TPA which is regarded as being a ma rke r of

proliferation, is a mixture of proteolytic f ragments conta in ing the relatively s table a -he l ica l rod

doma ins of s imple ep i the l ium type cytokerat ins . These f ragments are probably re leased du r ing necrosis a n d lysis of the ca rc inoma cells. Thus, TPA should be regarded as a broad s p e c t r u m epithelial t u m o r marke r and not as a specific molecular marke r for epithelial neop lasms .

I n t r a c e l l u l a r t u m o r m a r k e r s

In te rmedia te f i laments (IF) are a group of in t race l lu la r proteins which form the cytoskele ta l

network. They are 8-I 0 n m in d iameter and cons is t of about 5-6 cons t i t uen t prote ins which are

expressed in a highly t i ssue specific m a n n e r (57). During ma l ignan t t r a n s f o r m a t i o n their

express ion r emains t i s sue specific a l though there may be noticeable a l tera t ions in the i r pa t t e rn of express ion w h e n compared to the normal t issue (3). This proper ty of IF has been u s e d to

different iate the origin of carc inomas (223). Below (in Table 2) are descr ibed var ious in t race l lu lar

prote ins a n d the t i ssue for which they are character is t ic and can be u sed as ma rke r s

C o n s t i t u t e n t s o f IF

Cytokera t ins

Desmin

Viment in Neurof i laments a n d nes t in

Glial fibrillary acidic protein

K e r a t i n s a s t u m o r m a r k e r s

T a b l e 2

S p e c i f i c i t y

Tumors derived f rom epithelial cells

Tumors derived from musc le cells

Tumors derived from m e s e n c h y m a l cells Tumors derived from n e u r o n a l cells

Tumors derived from as t rocytes a n d glial cells

All type of epithelial cells con ta in 30-85% ofke ra t in s as the major cytoskele ta l protein. This

has led to exploring the possible use of kerat ins and its cons t i tuen t polypept ides as ma rke r s for

epithelioid mal ignanc ies (3).

Li tera ture su rvey reveals t ha t kera t in has been used effectively as a m a r k e r for epi thel ial

t u m o r s (for recent review; see Ref. 3), especially those of stratif ied a n d s q u a m o u s cell origin. For example lung ca rc inoma (37, 58-60), breas t carc inomas (61-63), u r ina ry b ladder ca rc inomas (64),

t h y m o m a s (65) and cervical carc inomas (66). Since the gas t ro in tes t ina l (GI) t rac t hn ing is of

epithelial origin, kera t in serves as a useful marker for GI tumors (48,67). Right f rom the buccal

cavity to the rec tum, so also pancreas and gall bladder are covered by epi thel ial l ining which

enables the special men t ion of kera t in as a marker in these cases. Oral cancers are mos t ly (90%)

of s q u a m o u s epithelial origin arid hence kera t ins are the best possible histological as well as

b iochemical ma rke r s for these tumors (68,69). Oesophagea lca rc inomas are also of epi thel ial origin

and m a n y workers have used kera t in to d is t inguish t h e m (70. Kerat in has also been widely u sed

as a ma rke r in prostat ic tumors (71, 72). Svanholm et. al. (73) have used ke ra t in to differentiate

be tween prostat ic hyperp las ia (a benign disorder) from prostat ic adenoca rc inoma .

VOL 7{2) In t race l lu la r Proteins as T u m o r Markers 85

We have used ke ra t in polypept ides as marke r s for gas t ro in tes t ina l c a r c i n o m a s a n d found

that in all the cases (barr ing s q u a m o u s cell c a r c inomas of the o e s o p h a g u s ) t he e x p r e s s i o n of

keratin in the ma l ignan t t i s sue dec reases when compared to n o r m a l t issue. We have found t ha t

normal colorecta l ep i t he l i um expresses two ke ra t in polypept ides whe reas the ma l ignan t l y

t ransformed cells express only a single polypept ide band (3]. Similarly, the n o r m a l gas t r ic

epithelium expreses four ke ra t i n polypept ides whereas the ma l ignan t t i s sue e x p r e s s e s two bands .

The a de noc a rc i nom as of the oesophgus show a m a r k e d r educ t ion in the e x p r e s s i o n of k e r a t i n

polypeptides w h e n c o m p a r e d wi th no rma l oesophagea l ep i the l ium which exp re s se s e ight k e r a t i n

polypeptides.

We have c o n c l u d e d in o u r s tudies tha t in the case of kera t ins , the a b s e n c e of a pa r t i cu l a r

keratin band c a n be u s e d as a m a r k e r in epithelioid gas t ro in tes t ina l mal ignanc ies . This is in to ta l

contrast to the p a t t e r n observed in o the r t u m o r marke r s so far. S e r u m t u m o r m a r k e r s show

significantly h igh levels in c a n c e r o u s condi t ions , whereas ma l ignan t t r a n s f o r m a t i o n o f ep i the l ia l

cells causes a d e c r e a s e d express ion of ke ra t in by a m e c h a n i s m ye t u n k n o w n .

In add i t ion to ke ra t ins , o the r cytoskele ta l pro te ins (21-23), oncogene r egu la to ry enzymes ,

enzymes involved in pos t - t r ans l a t i ona l modif icat ion of p ro te ins (24-27) a n d r ecep to r s c a n also

serve as t u m o r m a r k e r s . Bu t very little efforts have been made in this d i rec t ion to u se t he se

subs t ances in de t ec t ion of cance r s in h u m a n s in clinical b iochemis t ry labora tor ies . Never the less ,

efforts are be ing m a d e to simplify the t echn iques for thei r de tec t ion so they c a n be a d o p t e d in

clinical b ioc hemi s t r y labora tor ies .

C o n c l u s i o n

With the deve l opm en t of new and sensi t ive t echn iques for de tec t ion of very m i n u t e quan t i t i e s

of b iomolecules now it is poss ible to identify the changes in the levels o f g e n e r egu la to ry e n z y m e s

and o the r s u b s t a n c e s . Silver s ta in ing in SDS-PAGE ( 14), Radio r ecep to r [RRA] a n d Radio i m m u n o

assays [RIAl (75) enzym e l inked i u m m u n o assays [ELISA] (76), Western; N o r t h e r n a n d S o u t h e r n

blot t ing t e c h n i q u e s (77,78) a m o n g o thers c an de tec t up to p icogram quant i t i es . Cel lu lar p ro te ins

s u c h as cy toske le ta l prote ins , m e m b r a n e b o u n d pro te ins (37), c h r o m a t i n a s soc i a t ed p ro te ins a n d

gene r egu la to ry enzymes which are no t soluble in o rd ina ry a q u e o u s buffers a n d a re n o n - s e c r e t o r y

in n a t u r e c a n now be de t ec t ed in needle biopsies by the above m e n t i o n e d t echn iques . S tero id

ho rmone r ecep to r a s s ays are rou t ine ly done in m a n y clinical b iochemis t ry l abora to r ies in o rde r

to decide w h e t h e r the t u m o r f rom breas t , cervix or u t e ru s are h o r m o n e d e p e n d e n t or not .

There m a y be e i the r a n up regula t ion or down regula t ion of in t raceUular p ro te ins d u r i n g

mal ignan t t r a n s f o r m a t i o n depend i ng on the s t imu lus given a n d the t a rge t t i s sue involved (3).

Some pro te ins wh ich a re syn thes i zed in t u m o r cells and sec re t ed in the s e r u m a re u s e d for

de tec t ion a nd mon i to r ing the t u m o r activity. The o the r p ro te ins which a re no t s ec r e t ed c a n also

be u s e d as t u m o r m a r k e r s as descr ibed in this review. In t race l lu lar t u m o r m a r k e r s have a n

advan tage over conven t iona l t u m o r m arke r s in t ha t they c a n give va luab le i n f o r m a t i o n a b o u t the

origin a nd degree of d i f ferent ia t ion of tumors . There are var ious factors wh ich regu la te the

express ion of in t r ace l lu la r pro te ins , for example, down regula t ion o f k e r a t i n po lypep t ides is s e e n

in GI t u m o r s . The m e c h a n i s m of this up or down regula t ion is, however, no t k n o w n yet. More work

on gene regu la to rs will give be t te r insight for cance r de tec t ion a n d m a n a g e m e n t .

86 GUPTA ET AL. 1992

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